Hla-dr binding peptides and their uses

ABSTRACT

The present invention provides HLA-DR (MHC class II) binding peptides derived from the ovarian/breast cancer associated antigens, Human Epidermal Growth Factor Receptor 2 (HER-2/neu), Carcinoembryonic Antigen (CEA), Insulin Growth Factor Binding Protein 2 (IGFBP-2), and Cyclin D1. The immunogenic peptides can be used in cancer vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 60/984,646, filed on Nov. 1, 2007.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersCA107590 and CA015083 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to compositions and methods forpreventing, treating or diagnosing a number of pathological states suchas cancers. In particular, it provides novel peptides capable of bindingselected major histocompatibility complex (MHC) molecules and induce animmune response.

MHC molecules are classified as either Class I or Class II molecules.Class II MHC molecules are expressed primarily on cells involved ininitiating and sustaining immune responses, such as T lymphocytes, Blymphocytes, dendritic cells, macrophages, etc. Class II MHC moleculesare recognized by helper T lymphocytes and induce proliferation ofhelper T lymphocytes and amplification of the immune response to theparticular immunogenic peptide that is displayed. Complexes between aparticular disease-associated antigenic peptide and class II HLAmolecules are recognized by helper T lymphocytes and induceproliferation of helper T lymphocytes and amplification of specific CTLand antibody immune responses.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993).

Peptides of the present invention comprise epitopes that bind to HLAclass II DR molecules. A greater degree of heterogeneity in both sizeand binding frame position of the motif, relative to the N- andC-termini of the peptide, exists for class II peptide ligands. Thisincreased heterogeneity of HLA class II peptide ligands is due to thestructure of the binding groove of the HLA class II molecule which,unlike its class I counterpart, is open at both ends. Crystallographicanalysis of HLA class II DRB*0101-peptide complexes showed that themajor energy of binding is contributed by peptide residues complexedwith complementary pockets on the DRB*0101 molecules. An importantanchor residue engages the deepest hydrophobic pocket (see, e.g.,Madden, D. R. Ann. Rev. Immunol. 13:587, 1995) and is referred to asposition 1 (P1). P1 may represent the N-terminal residue of a class IIbinding peptide epitope, but more typically is flanked towards theN-terminus by one or more residues. Other studies have also pointed toan important role for the peptide residue in the sixth position towardsthe C-terminus, relative to P1, for binding to various DR molecules.

In the past few years evidence has accumulated to demonstrate that alarge fraction of HLA class I and class II molecules can be classifiedinto a relatively few supertypes, each characterized by largelyoverlapping peptide binding repertoires, and consensus structures of themain peptide binding pockets. Thus, peptides of the present inventionare identified by any one of several HLA-specific amino acid motifs, orif the presence of the motif corresponds to the ability to bind severalallele-specific HLA molecules, a supermotif. The HLA molecules that bindto peptides that possess a particular amino acid supermotif arecollectively referred to as an HLA “supertype.” Because human populationgroups, including racial and ethnic groups, have distinct patterns ofdistribution of HLA alleles it will be of value to identify motifs thatdescribe peptides capable of binding more than one HLA allele, so as toachieve sufficient coverage of all population groups. The presentinvention addresses these and other needs.

T lymphocytes recognize an antigen in the form of a peptide fragmentbound to the MHC class I or class II molecule rather than the intactforeign antigen itself Antigens presented by MHC class II molecules areusually soluble antigens that enter the antigen presenting cell viaphagocytosis, pinocytosis, or receptor-mediated endocytosis. Once in thecell, the antigen is partially degraded by acid-dependent proteases inendosomes. The resulting fragments or peptide associate with the MHCclass II molecule after the release of the CLIP fragment to form astable complex that is then transported to the surface for potentialrecognition by specific HTLs. See Blum, et al., Crit. Rev. Immunol., 17:411-17 (1997); Arndt, et al., Immunol. Res., 16: 261-72 (1997).

Peptides that bind a particular MHC allele frequently will fit within amotif and have amino acid residues with particular biochemicalproperties at specific positions within the peptide. Such residues areusually dictated by the biochemical properties of the MHC allele.Peptide sequence motifs have been utilized to screen peptides capable ofbinding MHC molecules (Sette, et al., Proc. Nati. Acad. Sci. USA 86:3296(1989)), and it has previously been reported that class I binding motifsidentified potential immunogenic peptides in animal models (De Bruijn,et al., Eur. J. Immunol. 21: 2963-70 (1991); Pamer, et al., Nature 353:852-955 (1991)). Also, binding of a particular peptide to a MHC moleculehas been correlated with immunogenicity of that peptide (Schaeffer, etal., Proc. Natl. Acad. Sci. USA 86:4649 (1989)).

Accordingly, while some MHC binding peptides have been identified, thereis a need in the art to identify novel MHC binding peptides from tumorassociated antigens that can be utilized to generate an immune responsein vaccines against these targets. Further, there is a need in the artto identify peptides capable of binding a wide array of different typesof MHC molecules such they are immunogenic in a large fraction a humanoutbred population.

One of the most formidable obstacles to the development of broadlyefficacious peptide-based immunotherapeutics has been the extremepolymorphism of HLA molecules. Effective coverage of a populationwithout bias would thus be a task of considerable complexity if epitopeswere used specific for HLA molecules corresponding to each individualallele because a huge number of them would have to be used in order tocover an ethnically diverse population. There exists, therefore, a needto develop peptide epitopes that are bound by multiple HLA antigenmolecules at high affinity for use in epitope-based vaccines. Thegreater the number of HLA antigen molecules bound, the greater thebreadth of population coverage by the vaccine. Analog peptides may beengineered based on the information disclosed herein and thereby used toachieve such an enhancement in breadth of population coverage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the identification of preexistent immunity to promiscuousHER-2/neu HLA-DR epitopes. Each panel shows a scatter gram of the meannumbers of T cells specific for one of the identified HER-2/neu (seeheader of each panel) peptides in both healthy volunteer donors andpatients. Each panel represents a unique peptide and each data point isderived from one individual. The grey box (i.e. cutoff) delineates theregion that constitutes the mean and two standard deviations calculatedfrom the normal healthy individuals. The percentages represent thefraction of patients that had mean T cell values above the cutoff. Thepeptides that are circled are those in which a higher fraction of thepatients responded compared to the normal healthy controls. Thesepeptides are considered to be the best vaccine candidates. However, itis clear that the rigidness of this approach could potentially result inmany false negatives. For example, while p885 was shown to be recognizedby 25% of patients, p886, a nearly identical peptide was found to berecognized by only 4%. However, if one looks at the graph for p886,there is a healthy individual that showed a robust response. Theexclusion of that data point would have resulted in a patient responserate of 15%, which would have been consistent with the p885 response.Despite this, we identified 5 candidates to move forward. The fidelityof this approach is evident from a prior study which has already shownthat a HER-2/neu peptide, p884-899, which encompasses the binding motifof p885, is an HLA-DR4 epitope.

FIG. 2 shows the identification of preexistent immunity to promiscuousCEA HLA-DR epitopes. FIG. 2 is identical to FIG. 1 with the onlyexception that CEA is the antigen. Seven candidate peptides wereidentified.

FIG. 3 shows the identification of preexistent immunity to promiscuousIGFBP2 HLA-DR epitopes. FIG. 3 is identical to FIG. 1 with the onlyexception that IGFBP2 is the antigen. Four candidate peptides wereidentified. Note that only 10 peptides were assessed as explained in thetext above.

FIG. 4 shows the identification of preexistent immunity to promiscuousIGFBP2 HLA-DR epitopes. FIG. 4 is identical to FIG. 1 with the onlyexception that Cyclin D1 is the antigen. Using the more liberalstatistical method, 7 potential epitopes were identified.

FIG. 5 shows the that HER-2/neu peptides, p59, p83, p88 and p885 arenaturally processed and presented antigens. IFN-γ ELISpot analysis ofshort term T cell lines generated against HER-2/neu peptides, p53 (PanelA), p83 (Panel B), p88 (Panel C) and p885 (Panel D). The lines weretested for responses against respective culture peptides, an irrelevant15-mer peptide, a HER-2/neu protein fragment (amino acids 22-122, PanelsA-C; amino acids 676-1255, Panel D), or an irrelevant similar weightprotein, ovalbumin. Each shows the results from two lines establishedfrom two different breast or ovarian cancer patients who had a positiveELlspot response to the peptide. Each bar is the mean (s.e.m.) of threereplicates.

FIG. 6 shows that IGFBP2 peptides p17, p22, p249, and p293 are naturallyprocessed peptides. FIG. 6 is identical to FIG. 5 except that theresults were obtained using IGFBP-2 derived helper epitopes.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to compositions and methods forpreventing, treating or diagnosing a number of pathological states suchas viral diseases and cancers. Thus, provided herein are novel peptidescapable of binding selected major histocompatibility complex (MHC)molecules and inducing or modulating an immune response. Some of thepeptides disclosed are capable of binding human class II MHC (HLA)molecules, including HLA-DR and HLA-DQ alleles. Also provided arecompositions that include immunogenic peptides having binding motifsspecific for MHC molecules. The peptides and compositions disclosed canbe utilized in methods for inducing an immune response, a helper Tlymphocyte (HTL) response, or a cytotoxic T lymphocyte (CTL) responsewhen administered to a system.

Epitopes on a number of immunogenic tumor associated antigens have beenidentified. The peptides are thus useful in pharmaceutical compositionsfor both in vivo and ex vivo therapeutic and diagnostic applications(e.g., tetramer reagents; Beckman Coulter).

The peptides are also useful as epitope-based vaccines. Theepitope-based vaccines preferably have enhanced, typically broadened,population coverage. The HLA-DR supermotif-bearing epitopes comprisingthe vaccine composition preferably bind to more than one HLA DRsupertype molecule with a KD of less than 1000 nM or 500 nM, andstimulate a HTL response in patients bearing an HLA DR supertype alleleto which the peptide binds.

Motif-bearing peptides may additionally be used as diagnostic, ratherthan immunogenic, reagents to evaluate an immune response. For example,an HLA-DR supermotif-bearing peptide epitope may be used prognosticallyto analyze an immune response for the presence of specific HTLpopulations from patients who possess an HLA DR supertype allele boundby the peptide epitope.

The binding affinity of a peptide epitope in accordance with theinvention for at least one HLA DR supertype molecule is preferablydetermined A preferred peptide epitope has a binding affinity of lessthan 1000 nM, or more preferably less than 500 nM for the at least oneHLA DR supertype molecule, and most preferably less than 50 nM.

Synthesis of a HLA DR supermotif-containing epitope may occur in vitroor in vivo. In a preferred embodiment, the peptide is encoded by arecombinant nucleic acid and expressed in a cell. The nucleic acid mayencode one or more peptides, at least one of which is an epitope of theinvention.

A peptide epitope of the invention, in the context of an HLA DRsupertype molecule to which it binds, can be contacted, either in vitroor in vivo, with a cytotoxic T lymphocyte and thereby be used to elicita T cell response in an HLA-diverse population.

An HTL epitope may be comprised by a single peptide. Further, the HTLepitope may be lipidated, preferably with palmitic acid, and may belinked by a spacer molecule to another HTL epitope or a CTL epitope. Theepitope may be expressed by a nucleotide sequence; in a preferredembodiment the nucleotide sequence is comprised in an attenuated viralhost.

As will be apparent from the discussion below, other embodiments ofmethods and compositions are also within the scope of the invention.Further, novel synthetic peptides produced by any of the methodsdescribed herein are also part of the invention.

The present invention provides peptides and nucleic acids encoding themfor use in vaccines and therapeutics. The invention provides methods ofinducing a helper T cell response against a preselected antigen in, apatient, the method comprising contacting a helper T cell with animmunogenic peptide of the invention. The peptides of the invention maybe derived from a number of tumor associated antigens. The methods ofthe invention can be carried out in vitro or in vivo. In a preferredembodiment the peptides are contacted with the helper T cell byadministering to the patient a nucleic acid molecule comprising asequence encoding the immunogenic peptide.

The present invention is directed to methods of modulating the bindingof peptide epitopes to HLA class II molecules. The invention includes amethod of modifying binding of an original peptide epitope that bears amotif correlated with binding to an HLA molecule, said motif comprisingat least one primary anchor position, said at least one primary anchorposition having specified therefore primary anchor amino acid residuesconsisting essentially of two or more residues, said method comprisingexchanging the primary anchor residue of the original peptide epitopefor another primary anchor residue, with the proviso that the originalprimary anchor residue is not the same as the exchanged primary anchorresidue. A preferred embodiment of the invention includes a method wherethe original primary anchor residue is a less preferred residue, and theexchanged residue is a more preferred residue.

One alternative embodiment of the invention includes a method ofmodifying binding of an original peptide epitope that bears a motifcorrelated with binding to an HLA molecule, said motif comprising atleast one primary anchor position having specified therefore at leastone primary anchor residue, and at least one secondary anchor positionhaving specified therefore at least one secondary residue, said methodcomprising exchanging the secondary anchor residue of the originalpeptide epitope for another secondary anchor residue, with the provisothat the original secondary anchor residue is different than theexchanged amino acid residue. In some cases the original secondaryresidue is a deleterious residue and the exchanged residue is a residueother than a deleterious residue and/or the original secondary anchorresidue is a less preferred residue and the exchanged residue is a morepreferred residue.

As will be apparent from the discussion below, other methods andembodiments are also contemplated. Further, novel synthetic peptidesproduced by any of the methods described herein are also part of theinvention.

Definitions

The following definitions are provided to enable one of ordinary skillin the art to understand some of the preferred embodiments of inventiondisclosed herein. It is understood, however, that these definitions areexemplary only and should not be used to limit the scope of theinvention as set forth in the claims. Those of ordinary skill in the artwill be able to construct slight modifications to the definitions belowand utilize such modified definitions to understand and practice theinvention disclosed herein. Such modifications, which would be obviousto one of ordinary skill in the art, as they may be applicable to theclaims set forth below, are considered to be within the scope of thepresent invention. If a definition set forth in this section is contraryto or otherwise inconsistent with a definition set forth in patents,published patent applications and other publications and sequences fromGenBank and other databases that are herein incorporated by reference,the definition set forth in this section prevails over the definitionthat is incorporated herein by reference.

An “HLA supertype or family”, as used herein, describes sets of HLAmolecules grouped on the basis of shared peptide-binding specificities,rather than serologic supertypes based on shared antigenic determinants.HLA class II molecules that share somewhat similar binding affinity forpeptides bearing certain amino acid motifs are grouped into HLAsupertypes. The terms “HLA superfamily,” “HLA supertype family,” “HLAfamily,” and “HLA xx-like molecules” (where xx denotes a particular HLAtype), are synonyms.

As used herein, the term “IC₅₀” refers to the concentration of peptidein a binding assay at which 50% inhibition of binding of a referencepeptide is observed. Depending on the conditions in which the assays arerun (i.e., limiting MHC proteins and labeled peptide concentrations),these values may approximate KD values. It should be noted that IC₅₀values can change, often dramatically, if the assay conditions arevaried, and depending on the particular reagents used (e.g., HLApreparation, etc.). For example, excessive concentrations of HLAmolecules will increase the apparent measured IC50 of a given ligand.

Alternatively, binding is expressed relative to a reference peptide. Asa particular assay becomes more, or less, sensitive, the IC₅₀'s of thepeptides tested may change somewhat. However, the binding relative tothe reference peptide will not change. For example, in an assay rununder conditions such that the IC₅₀ of the reference peptide increases10-fold, the IC₅₀ values of the test peptides will also shiftapproximately 10-fold. Therefore, to avoid ambiguities, the assessmentof whether a peptide is a good, intermediate, weak, or negative binderis generally based on its IC₅₀, relative to the IC₅₀ of a standardpeptide.

As used herein, “high affinity” with respect to peptide binding to HLAclass II molecules is defined as binding with an K_(D) (or IC₅₀) of lessthan 50 nM. “Intermediate affinity” is binding with a KD (or IC₅₀) ofbetween about 50 and about 500 nM. As used herein, “high affinity” withrespect to binding to HLA class II molecules is defined as binding withan KD (or IC₅₀) of less than 100 nM. “Intermediate affinity” is bindingwith a KD (or IC₅₀) of between about 100 and about 1000 nM. Assays fordetermining binding are described in detail, e.g., in PCT publicationsWO 94/20127 and WO 94/03205.

Binding may also be determined using other assay systems including thoseusing: live cells (e.g., Ceppellini et al., Nature 339:392 (1989);Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol.2:443 (1990); Hill et al.., J Immunol. 147:189 (1991); del Guercio etal., J Immunol. 154:685 (1995)), cell free systems using detergentlysates (e.g., Cerundolo et al., J Immunol. 21 :2069 (1991)),immobilized purified MHC (e.g., Hill et al., J Immunol. 152,2890 (1994);Marshall et al., J Immunol. 152:4946 (1994)), ELISA systems (e.g., Reayet al., EMBO J 11 :2829 (1992)), surface plasmon resonance (e.g., Khilkoet al., J Biol. Chem. 268:15425 (1993)); high flux soluble phase assays(Hammer et al., J. Exp. Med. 180:2353 (1994)).

The term “peptide” is used interchangeably with “oligopeptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of adjacent amino acids. Incertain embodiments, the oligopeptides of the invention are less thanabout 50 residues in length and usually consist of between about 6 andabout 25 residues, preferably 14 or 15 residues. Further, anoligopeptide of the invention can be such that it does not comprise morethan 50 contiguous amino acids of a native antigen. The preferredHTL-inducing peptides of the invention are 30 residues or less inlength, sometimes 20 residues or less and usually consist of betweenabout 6 and about 25 residues, preferably 14 or 15 residues.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

The nomenclature used to describe peptide compounds follows theconventional practice wherein the amino group is presented to the left(the N-terminus) and the carboxyl group to the right (the C-terminus) ofeach amino acid residue. In the formulae representing selected specificembodiments of the present invention, the amino- and carboxyl-terminalgroups, although not specifically shown, are in the form they wouldassume at physiologic pH values, unless otherwise specified. In theamino acid structure formula, each residue is generally represented bystandard three letter or single letter designations. The L-form of anamino acid residue is represented by a capital single letter or acapital first letter of a three-letter symbol, and the D-form for thoseamino acids having D-forms is represented by a lower case single letteror a lower case three letter symbol. Glycine has no asymmetric carbonatom and is simply referred to as “Gly” or G. Symbols for each aminoacids are shown below:

TABLE 1 Amino acids with their abbreviations Three letter Single letterAmino acid code code Alanine Ala A Arginine Arg R Asparagine Asn NAspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu EGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vivo or in vitro, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or HLA molecule. Throughout this disclosure epitope andpeptide are often used interchangeably.

It is to be appreciated that protein or peptide molecules that comprisean epitope of the invention as well as additional amino acid(s) arestill within the bounds of the invention. In certain embodiments, thereis a limitation on the length of a peptide of the invention. Theembodiment that is length-limited occurs when the protein/peptidecomprising an epitope of the invention comprises a region (i.e., acontiguous series of amino acids) having 100% identity with a nativesequence. In order to avoid the definition of epitope from reading,e.g., on whole natural molecules, there is a limitation on the length ofany region that has 100% identity with a native peptide sequence. Thus,for a peptide comprising an epitope of the invention and a region with100% identity with a native peptide sequence, the region with 100%identity to a native sequence generally has a length of: less than orequal to 600 amino acids, often less than or equal to 500 amino acids,often less than or equal to 400 amino acids, often less than or equal to250 amino acids, often less than or equal to 100 amino acids; often lessthan or equal to 85 amino acids, often less than or equal to 75 aminoacids, often less than or equal to 65 amino acids, and often less thanor equal to 50 amino acids. In certain embodiments, an “epitope” of theinvention is comprised by a peptide having a region with less than 51amino acids that has 100% identity to a native peptide sequence, in anyincrement down to 5 amino acids.

Accordingly, peptide or protein sequences longer than 600 amino acidsare within the scope of the invention, so long as they do not compriseany contiguous sequence of more than 600 amino acids that have 100%identity with a native peptide sequence. For any peptide that has fivecontiguous residues or less that correspond to a native sequence, thereis no limitation on the maximal length of that peptide in order to fallwithin the scope of the invention. It is presently preferred that a CTLepitope be less than 600 residues long in any increment down to eightamino acid residues.

A “dominant epitope” induces an immune response upon immunization withwhole native antigens which comprise the epitope. (See, e.g., Sercarz,et al., Annu. Rev. Immunol. 11:729-766 (1993)). Such a response iscross-reactive in vitro with an isolated peptide epitope.

A “cryptic epitope” elicits a response by immunization with isolatedpeptide, but the response is not cross-reactive in vitro when intactwhole protein which comprises the epitope is used as an antigen.

A “subdominant epitope” is an epitope which evokes little or no responseupon immunization with whole antigens which comprise the epitope, butfor which a response can be obtained by immunization in vivo or in vitrowith an isolated epitope, and this response (unlike the case of crypticepitopes) is detected when whole protein is used to recall the responsein vitro.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservatives, and the like.

As used herein, the term “pharmaceutically acceptable” refers to agenerally non-toxic, inert, and/or physiologically compatiblecomposition.

As used herein, the term “protective immune response” or “therapeuticimmune response” refers to a HTL and/or a CTL response to a tumorassociated antigen, which in some way prevents or at least partiallyarrests disease symptoms, side effects or progression. The immuneresponse may include an antibody response that has been facilitated bythe stimulation of helper T cells.

In certain embodiments, an “immunogenic peptide” is a peptide whichcomprises an allele-specific motif such that the peptide will bind anMHC (HLA) molecule and induce a HTL response Immunogenic peptides of theinvention are capable of binding to an appropriate class II MHC molecule(e.g., HLA-DR) and inducing a helper T cell response against the antigenfrom which the immunogenic peptide is derived.

An “immunogenic response” includes one that stimulates a HTL and/or CTLresponse in vitro and/or in vivo as well as modulates an ongoing immuneresponse through directed induction of cell death (or apoptosis) inspecific T cell populations.

Immunogenic peptides of the invention are capable of binding to anappropriate HLA-DR molecule and inducing a helper T-cell responseagainst the antigen from which the immunogenic peptide is derived. Theimmunogenic peptides of the invention are less than about 50 residues inlength, often 30 residues or less in length, or 20 residues or less inlength and usually consist of between about 6 and about 25 residues,preferably 14 or 15 residues.

The term “derived” when used to discuss an epitope is a synonym for“prepared.” A derived epitope can be isolated from a natural source, orit can be synthesized in accordance with standard protocols in the art.Synthetic epitopes can comprise artificial amino acids “amino acidmimetics,” such as D isomers of natural occurring L amino acids ornon-natural amino acids such as cyclohexylalanine. A derived/preparedepitope can be an analog of a native epitope.

Immunogenic peptides are conveniently identified using the binding motifalgorithms described for the specific HLA subtype (e.g., HLA-DR). Thealgorithms are mathematical procedures that produce a score whichenables the selection of immunogenic peptides. Typically one uses thealgorithmic score with a “binding threshold” to enable selection ofpeptides that have a high probability of binding at a certain affinityand will in turn be immunogenic. The algorithm is based upon either theeffects on MHC binding of a particular amino acid at a particularposition of a peptide or the effects on binding of a particularsubstitution in a motif containing peptide.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into an oligopeptide by an amide bond or amide bondmimetic.

A “conserved residue” is an amino acid which occurs in a significantlyhigher frequency than would be expected by random distribution at aparticular position in a peptide. Typically a conserved residue is onewhere the MHC structure may provide a contact point with the immunogenicpeptide. At least one to three or more, preferably two, conservedresidues within a peptide of defined length defines a motif for animmunogenic peptide. These residues are typically in close contact withthe peptide binding groove, with their side chains buried in specificpockets of the groove itself Typically, an immunogenic peptide willcomprise up to three conserved residues, more usually two conservedresidues.

The term “motif” refers to the pattern of residues in a peptide ofdefined length, usually about 6 to about 25 amino acids, which isrecognized by a particular MHC allele (one or more HLA molecules). Thepeptide motifs are typically different for each human MHC allele anddiffer in the pattern of the highly conserved residues and negativeresidues. Peptide motifs are often unique for the protein encoded byeach human HLA allele, differing in their pattern of the primary andsecondary anchor residues. Typically as used herein, a “motif” refers tothat pattern of residues which is recognized by an HLA molecule encodedby a particular allele. The binding motif for an allele can be definedwith increasing degrees of precision.

The designation of a residue position in an epitope as the “carboxylterminus” or the “carboxyl terminal position” refers to the residueposition at the end of the epitope which is nearest to the carboxylterminus of a peptide, which is designated using conventionalnomenclature as defined below. The “carboxyl terminal position” of theepitope may or may not actually correspond to the end of the peptide orpolypeptide.

The designation of a residue position in an epitope as “amino terminus”or “amino-terminal position” refers to the residue position at the endof the epitope which is nearest to the amino terminus of a peptide,which is designated using conventional nomenclature as defined below.The “amino terminal position” of the epitope may or may not actuallycorrespond to the end of the peptide or polypeptide.

A “motif bearing peptide” or “peptide which comprises a motif” refers toa peptide that comprises primary anchors specified for a given motif orsupermotif.

In certain embodiments, a “supermotif” is a peptide binding specificityshared by HLA molecules encoded by two or more HLA alleles. Preferably,a supermotif-bearing peptide is recognized with high or intermediateaffinity (as defined herein) by two or more HLA molecules or antigens.

Alternatively, the term “supermotif” refers to motifs that, when presentin an immunogenic peptide, allow the peptide to bind more than one HLAantigen. The supermotif preferably is recognized with high orintermediate affinity (as defined herein) by at least one HLA allelehaving a wide distribution in the human population, preferablyrecognized by at least two alleles, more preferably recognized by atleast three alleles, and most preferably recognized by more than threealleles.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

“Major Histocompatibility Complex” or “MHC” is a cluster of genes whichplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press,New York, 1993.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. Thus, the peptides of thisinvention do not contain materials normally associated with their insitu environment, e.g., MHC class II molecules on antigen presentingcells. Even where a protein has been isolated to a homogenous ordominant band, there are trace contaminants in the range of 5-10% ofnative protein which co-purify with the desired protein. Isolatedpeptides of this invention do not contain such endogenous co-purifiedprotein.

“Peripheral blood mononuclear cells” (PBMCs) are cells found in from theperipheral blood of a patient. PBMCs comprise, e.g., CTLs and HTLs andantigen presenting cells. These cells can contact an antigen in vivo, orbe obtained from a mammalian source and contacted with an antigen invitro.

“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is degenerate binding.

“Promiscuous recognition” is where the same peptide bound by differentHLA molecules is recognized by the same T cell clone. It may also referto the ability of a peptide to be recognized by a single T cell receptorin the context of multiple HLA alleles.

“Link” or “join” refers to any method known in the art for functionallyconnecting peptides, including, without limitation, recombinant fusion,covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding,and electrostatic bonding.

A “non-native” sequence or “construct” refers to a sequence that is notfound in nature, i.e., is “non-naturally occurring”. Such sequencesinclude, e.g., peptides that are lipidated or otherwise modified, andpolyepitopic compositions that contain epitopes that are not contiguousin a native protein sequence.

As used herein, a “vaccine” is a composition that contains one or morepeptides of the invention, see, e.g., TABLE I. There are numerousembodiments of vaccines in accordance with the invention, such as by acocktail of one or more peptides; one or more peptides of the inventioncomprised by a polyepitopic peptide; or nucleic acids that encode suchpeptides or polypeptides, e.g., a minigene that encodes a polyepitopicpeptide. The “one or more peptides” can include any whole unit integerfrom 1-150, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or morepeptides of the invention. The peptides or polypeptides can optionallybe modified, such as by lipidation, addition of targeting or othersequences. HLA class II-binding peptides of the invention can be linkedto HLA class I-binding peptides, to facilitate activation of bothcytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprisepeptide pulsed antigen presenting cells, e.g., dendritic cells.

DETAILED DESCRIPTION OF THE INVENTION

Certain embodiments of the present invention relate in part to anepitope-based approach for vaccine design. Such an approach is based onthe well-established finding that the mechanism for inducing HTL immuneresponse comprises the step of presenting a HTL epitope as a peptide ofabout 6-25 amino acids bound to an HLA molecule displayed on anantigen-presenting cell.

Certain embodiments of the present invention relate to peptidescomprising allele-specific peptide motifs and supermotifs which bind toHLA class II molecules.

As noted above, high HLA binding affinity is correlated with higherimmunogenicity. Higher immunogenicity can be manifested in severaldifferent ways. For instance, a higher binding peptide will beimmunogenic more often. Close to 90% of high binding peptides areimmunogenic, as contrasted with about 50% of the peptides which bindwith intermediate affinity. A higher binding peptide will also lead to amore vigorous response. As a result, less peptide is required to elicita similar biological effect. Thus, in some embodiments of the inventionhigh binding epitopes are particularly desired.

It has been noted that a significant number of epitopes derived fromknown non-viral tumor associated antigens (TAA) bind HLA Class II withintermediate affinity (IC₅₀ in the 50-500 mM range). It has been foundthat 8 of 15 known TAA peptides recognized by tumor infiltratinglymphocytes (TIL) or CTL bound in the 50-500 mM range. These data are incontrast with estimates that 90% of known viral antigens that wererecognized as peptides bound HLA with IC₅₀ of 50 mM or less while onlyapproximately 10% bound in the 50-500 mM range (Sette, et al., J.Immunol., 153:5586-5592 (1994)). This phenomenon is probably due in thecancer setting to elimination, or functional inhibition of the CTLrecognizing several of the highest binding peptides, presumably becauseof T cell tolerization events.

Epitope-bearing peptides in accordance with the invention can beprepared synthetically, by recombinant DNA technology, or from naturalsources such as whole viruses or tumors. Although the peptide willpreferably be substantially free of other naturally occurring host cellproteins and fragments thereof, in some embodiments the peptides aresynthetically conjugated to native molecules or particles; the peptidescan also be conjugated to non-native molecules or particles.

The peptides in accordance with the invention can be a variety oflengths, and either in their neutral (uncharged) forms or in forms whichare salts. The peptides in accordance with the invention are either freeof modifications such as glycosylation, side chain oxidation, orphosphorylation; or they contain these modifications.

Desirably, the epitope-bearing peptide will be as small as possiblewhile still maintaining relevant immunologic activity of the largepeptide; of course it is particularly desirable with peptides frompathogenic organisms that the peptide be small in order to avoidpathogenic function. When possible, it may be desirable to optimizeepitopes of the invention to a length of about 6 to about 25, preferably14 to 15 amino acid residues for a class II molecule. Preferably, thepeptides are commensurate in size with endogenously processed viralpeptides or tumor cell peptides that are bound to HLA class I or classII molecules on the cell surface. Nevertheless, the identification andpreparation of peptides of other lengths can be carried out using thetechniques described here such as the disclosures of primary anchorpositions. It is to be appreciated that peptide epitopes in accordancewith the invention can be present in peptides or proteins that arelonger than the epitope itself Moreover, multiepitopic peptides cancomprise at least one epitope of the invention along with otherepitope(s).

In particular, the invention provides motifs that are common to peptidesbound by more than one HLA allele. By a combination of motifidentification and MHC-peptide interaction studies, peptides useful forpeptide vaccines have been identified.

Peptides comprising the epitopes from these antigens are synthesized andthen tested for their ability to bind to the appropriate MHC moleculesin assays using, for example, immunofluorescent staining and flowmicrofluorometry, peptide-dependent class II assembly assays. Thosepeptides that bind to the class II molecule are further evaluated fortheir ability to serve as targets for HTLs derived from infected orimmunized individuals, as well as for their capacity to induce primaryin vitro or in vivo HTL responses that can give rise to HTL populationscapable of reacting with tumor cells as potential therapeutic agents.

The starting point, therefore, for the design of effective vaccines isto ensure that the vaccine will generate a large number of epitopes thatcan successfully be presented. It may be possible to administer thepeptides representing the epitopes per se. Such administration isdependent on the presentation of “empty” HLA molecules displayed on thecells of the subject. In one approach to use of the immunogenic peptidesper se, these peptides may be incubated with antigen-presenting cellsfrom the subject to be treated ex vivo and the cells then returned tothe subject.

Alternatively, the peptides can be generated in situ by administering anucleic acid containing a nucleotide sequence encoding it. Means forproviding such nucleic acid molecules are described in WO99/58658, thedisclosure of which is incorporated herein by reference. Further, theimmunogenic peptides can be administered as portions of a larger peptidemolecule and cleaved to release the desired peptide. The larger peptidemay contain extraneous amino acids, in general the fewer the better.Thus, peptides which contain such amino acids are typically 50 aminoacids or less, more typically 30 amino acids or less, and more typically20 amino acids or less. The precursor may also be a heteropolymer orhomopolymer containing a multiplicity of different or same HTL epitopes.Of course, mixtures of peptides and nucleic acids which generate avariety of immunogenic peptides can also be employed. The design of thepeptide vaccines, the nucleic acid molecules, or the hetero- orhomo-polymers is dependent on the inclusion of the desired epitope.

In certain embodiments, it is preferred that peptides include an epitopethat binds to an HLA-DR supertype allele. These motifs may be used todefine T-cell epitopes from any desired antigen, particularly thoseassociated with human cancers for which the amino acid sequence of thepotential antigen targets is known.

The peptides are thus useful in pharmaceutical compositions for both invivo and ex vivo therapeutic and diagnostic applications.

Peptides comprising the supermotif sequences can be identified, as notedabove, by screening potential antigenic sources. Useful peptides canalso be identified by synthesizing peptides with systematic or randomsubstitution of the variable residues in the supermotif, and testingthem according to the assays provided. As demonstrated below, it isuseful to refer to the sequences of the target HLA molecule, as well.

For epitope-based vaccines, the peptides of the present inventionpreferably comprise a supermotif and/or motif recognized by an HLA classII molecule having a wide distribution in the human population. Thelarge degree of HLA polymorphism is an important factor to be taken intoaccount with the epitope-based approach to vaccine development. Toaddress this factor, epitope selection encompassing identification ofpeptides capable of binding at high or intermediate affinity to multipleHLA molecules is preferably utilized, most preferably these epitopesbind at high or intermediate affinity to two or more allele-specific HLAmolecules.

HTL-inducing peptides of interest for vaccine compositions preferablyinclude those that have an IC₅₀ or binding affinity value for class IIHLA molecules, 1000 nM or better (i.e., the value is greater than orequal to 1000 nM). For example, peptide binding is assessed by testingthe capacity of a candidate peptide to bind to a purified HLA moleculein vitro. Peptides exhibiting high or intermediate affinity are thenconsidered for further analysis. Selected peptides are generally testedon other members of the supertype family. In preferred embodiments,peptides that exhibit cross-reactive binding are then used in cellularscreening analyses or vaccines.

Definition of motifs that are predictive of binding to specific class IIalleles allows the identification of potential peptide epitopes from anantigenic protein whose amino acid sequence is known. Typically,identification of potential peptide epitopes is initially carried outusing a computer to scan the amino acid sequence of a desired antigenfor the presence of motifs and/or supermotifs.

The previous definition of motifs specific for different class IIalleles allows the identification of potential peptide epitopes from anantigenic protein whose amino acid sequence is known. Typically,identification of potential peptide epitopes is initially carried outusing a computer to scan the amino acid sequence of a desired antigenfor the presence of motifs. The epitopic sequences are then synthesized.The capacity to bind MHC Class II molecules is measured in a variety ofdifferent ways.

The procedures used to identify peptides of the present inventiongenerally follow the methods disclosed in Falk et al., Nature 351:290(1991), which is incorporated herein by reference. Briefly, the methodsinvolve large-scale isolation of MHC class II molecules, typically byimmunoprecipitation or affinity chromatography, from the appropriatecell or cell line. Examples of other methods for isolation of thedesired MHC molecule equally well known to the artisan include ionexchange chromatography, lectin chromatography, size exclusion, highperformance ligand chromatography, and a combination of all of the abovetechniques.

The peptides bound to the peptide binding groove of the isolated MHCmolecules are eluted typically using acid treatment. Peptides can alsobe dissociated from class II molecules by a variety of standarddenaturing means, such as heat, pH, detergents, salts, chaotropicagents, or a combination thereof

Peptide fractions are further separated from the MHC molecules byreversed-phase high performance liquid chromatography (HPLC) andsequenced. Peptides can be separated by a variety of other standardmeans well known to the artisan, including filtration, ultrafiltration,electrophoresis, size chromatography, precipitation with specificantibodies, ion exchange chromatography, isoelectrofocusing, and thelike.

Sequencing of the isolated peptides can be performed according tostandard techniques such as Edman degradation (Hunkapiller, M. W., etal., Methods Enzymol. 91, 399 [1983]). Other methods suitable forsequencing include mass spectrometry sequencing of individual peptidesas previously described (Hunt, et al., Science 225:1261 (1992), which isincorporated herein by reference) Amino acid sequencing of bulkheterogenous peptides (e.g., pooled HPLC fractions) from different classI molecules typically reveals a characteristic sequence motif for eachclass I allele.

Next, peptides that test positive in the MHC class II binding assay areassayed for the ability of the peptides to induce specific HTL responsesin vitro. For instance, antigen-presenting cells that have beenincubated with a peptide can be assayed for the ability to induce HTLresponses in responder cell populations. Antigen-presenting cells can benormal cells such as peripheral blood mononuclear cells or dendriticcells (Inaba, et al., J. Exp. Med. 166:182 (1987); Boog, Eur. J.Immunol, 18:219 (1988)).

As disclosed herein, higher HLA binding affinity is correlated withgreater immunogenicity. Greater immunogenicity can be manifested inseveral different ways. Immunogenicity can correspond to whether animmune response is elicited at all, and to the vigor of any particularresponse, as well as to the extent of a diverse population in which aresponse is elicited. For example, a peptide might elicit an immuneresponse in a diverse array of the population, yet in no instanceproduce a vigorous response. In accordance with the principles disclosedherein, close to 90% of high binding peptides have been found to beimmunogenic, as contrasted with about 50% of the peptides which bindwith intermediate affinity. Moreover, higher binding affinity peptideslead to more vigorous immunogenic responses. As a result, less peptideis required to elicit a similar biological effect if a high affinitybinding peptide is used. Thus, in preferred embodiments of theinvention, high affinity binding epitopes are particularly useful.Nevertheless, improvements over the prior art are achieved withintermediate or high binding peptides.

After determining their binding affinity, additional confirmatory workcan be performed to select, amongst these vaccine candidates, epitopeswith preferred characteristics in terms of population coverage,antigenicity, and immunogenicity.

Thus, various strategies can be utilized to evaluate immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998); This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In thismethod, peptides in incomplete Freund's adjuvant are administeredsubcutaneously to HLA transgenic mice. Several weeks followingimmunization, splenocytes are removed and cultured in vitro in thepresence of test peptide for approximately one week. Peptide-specific Tcells are detected.

3) Demonstration of recall T cell responses from patients who have beeneffectively vaccinated or who have a tumor; (see, e.g., Rehermann, B. etal., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97,1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S.C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J.Virol. 71:6011, 1997; Tsang et al., J. Natl. Cancer Inst. 87:982-990,1995; Disis et al., J Immunol. 156:3151-3158, 1996). In applying thisstrategy, recall responses are detected by culturing PBL from patientswith cancer who have generated an immune response “naturally”, or frompatients who were vaccinated with tumor antigen vaccines.

PBL from subjects are cultured in vitro for 1-2 weeks in the presence oftest peptide plus antigen presenting cells (APC) to allow activation of“memory” T cells, as compared to “naive” T cells. At the end of theculture period, T cell activity is detected.

An immunogenic peptide epitope of the invention may be included in apolyepitopic vaccine composition comprising additional peptide epitopesof the same antigen, antigens from the same source, and/or antigens froma different source. Moreover, class II epitopes can be included alongwith class I epitopes. Peptide epitopes from the same antigen may beadjacent epitopes that are contiguous in sequence or may be obtainedfrom different regions of the protein.

An epitope present in the peptides of the invention can becross-reactive or non-cross-reactive in its interactions with MHCalleles and allele subtypes. Cross-reactive binding of an epitope (orpeptide) permits an epitope to be bound by more than one HLA molecule.Such cross-reactivity is also known as degenerate binding. Anon-cross-reactive epitope would be restricted to binding a particularMHC allele or allele subtype.

Motifs Indicative of Class II HTL Inducing Peptide Epitope

The primary anchor residues of the HLA class II supermotifs and motifsare delineated below.

HLA DR-1-4-7 Supermotif

Motifs have also been identified for peptides that bind to three commonHLA class II allele-specific HLA molecules: HLA DRB1*0401, DRB1*0101,and DRB1*0701 (see, e.g., the review by Southwood et al. J. Immunology160:3363-3373,1998). Collectively, the common residues from these motifsdelineate the HLA DR-1-4-7 supermotif. Peptides that bind to these DRmolecules carry a supermotif characterized by a large aromatic orhydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residuein position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L,or M) as a primary anchor residue in position 6 of a 9-mer core region.Allele-specific secondary effects and secondary anchors for each ofthese HLA types have also been identified (Southwood et al., supra).Peptide binding to HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can bemodulated by substitutions at primary and/or secondary anchor positions,preferably choosing respective residues specified for the supermotif.

Two alternative motifs (i.e., submotifs) characterize peptide epitopesthat bind to HLA-DR3 molecules (see, e.g., Geluk et al., J. Immunol.152:5742, 1994). In the first motif (submotif DR3A) a large, hydrophobicresidue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mercore, and D is present as an anchor at position 4, towards the carboxylterminus of the epitope. As in other class II motifs, core position 1may or may not occupy the peptide N-terminal position.

The alternative DR3 submotif provides for lack of the large, hydrophobicresidue at anchor position 1, and/or lack of the negatively charged oramide-like anchor residue at position 4, by the presence of a positivecharge at position 6 towards the carboxyl terminus of the epitope. Thus,for the alternative allele-specific DR3 motif (submotif DR3B): L, I, V,M, F, Y, A, or Y is present at anchor position 1; D, N, Q, E, S, or T ispresent at anchor position 4; and K, R, or H is present at anchorposition 6. Peptide binding to HLA-DR3 can be modulated by substitutionsat primary and/or secondary anchor positions, preferably choosingrespective residues specified for the motif.

As with HLA class I binding peptides, motifs have also been defined forHLA class II-binding peptides. Several studies have identified animportant role for an aromatic or hydrophobic residue (I, L, M, V, F, W,or Y) at position 1 of a 9-mer core region, typically nested within alonger peptide sequence, in the binding of peptide ligands to severalHLA-class II alleles (Hammer et al. Cell 74:197, (1993); Sette et al. J.Immunol. 151:3163-70 (1993); O'Sullivan et al. J. Immunol. 147:2663(1991); and Southwood et al. J. Immunol. 160:3363-73 (1998)). A strongrole has also been demonstrated for the residue in position 6 of the9-mer core, where short and/or hydrophobic residues (S, T, C, A, P, V,I, L, or M) are preferred. This position 1-position 6 motif has beendescribed as a DR-supermotif (Southwood et al. J. Immunol. 160:3363-3373(1998)) and has been shown to efficiently identify peptides capable ofbinding a large set of common HLA-class II alleles.

Peptides binding to class II molecules may also be analyzed with respectto the identification of secondary preferred or deleterious residues.For example, to derive a more detailed DRB1*0401 motif to definesecondary residues influencing peptide binding, we employed a strategysimilar to that performed with class I peptides. For each peptideanalyzed, nine-residue-long core regions were aligned on the basis ofthe primary class II positions P1 and P6 anchors. Then, the averagebinding affinity of a peptide carrying a particular residue wascalculated for each position, relative to the remainder of the group.Following this method, values showing average relative binding werecompiled. These values also present a map of the positive or negativeeffect of each of the 20 naturally occurring amino acids in DRB1*0401binding capacity when occupying a particular position relative to theP1-P6 class II motif positions.

Variations in average relative binding of greater than or equal tofourfold or less than or equal to 0.25 were arbitrarily consideredsignificant and indicative of secondary effects of a given residue onHLA-peptide interactions. Most secondary effects were associated withP4, P7, and P9. These positions correspond to secondary anchors engagingshallow pockets on the DR molecule. Similar studies defining secondaryresidues were also performed for DRB1*0101 and DRB1*0701. Thedefinitions of secondary residues of motifs for DR1, DR4, and DR7 areshown in TABLE 139.

Upon definition of allele-specific secondary effects and secondaryanchors, allele-specific algorithms were derived and utilized toidentify peptides binding DRB1*0101, DRB1*0401, and DRB*0701. Furtherexperiments, identified a large set of HLA class II molecules, whichincludes at least the DRB1*0101, DRB1*0401, and DRB*0701, DRB1*1501,DRB1*0901 and DRB1*1302 allelic products recognizing the DR supermotif,and is characterized by largely overlapping peptide binding repertoires.

The data presented above confirm that several common HLA class II typesare characterized by largely overlapping peptide binding repertoires. Onthis basis, in analogy to the case of HLA class I molecules, HLA classII molecules can be grouped in a HLA class II supertype, defined andcharacterized by similar, or largely overlapping (albeit not identical)peptide binding specificities.

The peptides present in the invention can be identified by any suitablemethod. For example, peptides are conveniently identified using thealgorithms of the invention described in the co-pending U.S. patentapplication Ser. No. 09/894,018. These algorithms are mathematicalprocedures that produce a score which enables the selection ofimmunogenic peptides. Typically one uses the algorithmic score with abinding threshold to enable selection of peptides that have a highprobability of binding at a certain affinity and will in turn beimmunogenic. The algorithm are based upon either the effects on MHCbinding of a particular amino acid at a particular position of a peptideor the effects on binding MHC of a particular substitution in a motifcontaining peptide.

Peptide sequences characterized in molecular binding assays and captureassays have been and can be identified utilizing various technologies.Motif-positive sequences are identified using a customized applicationcreated at Epimmune. Sequences are also identified utilizingmatrix-based algorithms, and have been used in conjunction with a“power” module that generates a predicted 50% inhibitory concentration(PIC) value. These latter methods are operational on Epimmune'sHTML-based Epitope Information System (EIS) database. All of thedescribed methods are viable options in peptide sequence selection forIC₅₀ determination using binding assays.

The capacity to bind MHC molecules is measured in a variety of differentways. One means is a MHC binding assay as described in the relatedapplications, noted above. Other alternatives described in theliterature include inhibition of antigen presentation (Sette, et al., J.Immunol. 141:3893 (1991), in vitro assembly assays (Townsend, et al.,Cell 62:285 (1990), and FACS based assays using mutated cells, such asRMA.S (Melief, et al., Eur. J. Immunol. 21:2963 (1991)).

Capture Assay: Unlike the HPLC-based molecular binding assay, notedabove, the high throughput screening (“HTS”) Capture assay does notutilize a size-exclusion silica column for separation of bound fromunbound radioactive marker. Instead, wells of an opaque white 96-wellOptiplate (Packard) are coated with 3 μg (100 μl@ 30 μg/ml) ofHLA-specific antibody (Ab) that “capture” complexes of radiolabeled MHCand unlabeled peptide transferred from the molecular binding assay platein 100 μl of 0.05% NP40/PBS. After a 3-hour incubation period, thesupernatant is decanted and scintillation fluid (Microscint 20) added.Captured complexes are then measured on a microplate scintillation andluminescence counter (TopCount NXTTM; Packard).

Additional assays for determining binding are described in detail, i.e.,in PCT publications WO 94/20127 and WO 94/03205. Binding data resultsare often expressed in terms of IC₅₀ value. IC₅₀ is the concentration ofpeptide in a binding assay at which 50% inhibition of binding of areference peptide occurs. Given the conditions in which the assays arepreformed (i.e., limiting MHC proteins and labeled peptideconcentrations), these values approximate K_(D) values. It should benoted that IC₅₀ values can change, often dramatically, if the assayconditions are varied, and depending on the particular reagents used(i.e., MHC preparation, etc.). For example, excessive concentrations ofMHC molecules will increase the apparent measured IC₅₀ of a givenligand. Alternatively, binding is expressed relative to a referencepeptide. Although as a particular assay becomes more, or less,sensitive, the IC₅₀'s of the peptides tested may change somewhat, thebinding relative to the reference peptide will not significantly change.For example, in an assay preformed under conditions such that the IC₅₀of the reference peptide increases 10-fold, the IC₅₀ values of the testpeptides will also increase approximately 10-fold. Therefore, to avoidambiguities, the assessment of whether a peptide is a good,intermediate, weak, or negative binder is generally based on its IC₅₀,relative to the IC₅₀ of a standard peptide.

The peptides of the invention may also comprise isosteres of two or moreresidues in the MHC-binding peptide. An isostere as defined here is asequence of two or more residues that can be substituted for a secondsequence because the steric conformation of the first sequence fits abinding site specific for the second sequence. The term specificallyincludes peptide backbone modifications well known to those skilled inthe art. Such modifications include modifications of the amide nitrogen,the α-carbon, amide carbonyl, complete replacement of the amide bond,extensions, deletions or backbone crosslinks. See, generally, Spatola,Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.VII (Weinstein ed., 1983).

Modifications of peptides with various amino acid mimetics or unnaturalamino acids are particularly useful in increasing the stability of thepeptide in vivo. Stability can be assayed in a number of ways. Forinstance, peptidases and various biological media, such as human plasmaand serum, have been used to test stability. See, e.g., Verhoef et al.,Eur. J. Drug Metab. Pharmacokin. 11:291-302 (1986). Half life of thepeptides of the present invention is conveniently determined using a 25%human serum (v/v) assay. The protocol is generally as follows. Pooledhuman serum (Type AB, non-heat inactivated) is delipidated bycentrifugation before use. The serum is then diluted to 25% with RPMItissue culture media and used to test peptide stability. Atpredetermined time intervals a small amount of reaction solution isremoved and added to either 6% aqueous trichloracetic acid or ethanol.The cloudy reaction sample is cooled (4° C.) for 15 minutes and thenspun to pellet the precipitated serum proteins. The presence of thepeptides is then determined by reversed-phase HPLC usingstability-specific chromatography conditions.

Such analogs may also possess improved shelf-life or manufacturingproperties. More specifically, non-critical amino acids need not belimited to those naturally occurring in proteins, such as L-α-aminoacids, or their D-isomers, but may include non-natural amino acids aswell, such as amino acids mimetics, e.g. D- or L- naphylalanine; D- orL-phenylglycine; D- or L-2-thieneylalanine; D- or L-1,-2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluorophenylalanine; D- or L- p-biphenylphenylalanine; D- or L-p-methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D-or L-alkylalanines, where the alkyl group can be a substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromaticrings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl,pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings.

Another embodiment for generating effective peptide analogs involves thesubstitution of residues that have an adverse impact on peptidestability or solubility in, e.g., a liquid environment. Thissubstitution may occur at any position of the peptide epitope. Analogsof the present invention may include peptides containing substitutionsto modify the physical property (e.g., stability or solubility) of theresulting peptide. For example, a cysteine (C) can be substituted out infavor of α-amino butyric acid. Due to its chemical nature, cysteine hasthe propensity to form disulfide bridges and sufficiently alter thepeptide structurally so as to reduce binding capacity. Substitutingα-amino butyric acid for C not only alleviates this problem, butactually improves binding and crossbinding capability in certaininstances (see, e.g., the review by Sette et al., In: Persistent ViralInfections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England,1999). Substitution of cysteine with α-amino butyric acid may occur atany residue of a peptide epitope, i.e. at either anchor or non-anchorpositions.

The binding activity, particularly modification of binding affinity orcross-reactivity among HLA supertype family members, of peptides of theinvention can also be altered using analoging, which is described inco-pending U.S. application Ser. No. 09/226,775 filed Jan. 6, 1999. Inbrief, the analoging strategy utilizes the motifs or supermotifs thatcorrelate with binding to certain HLA molecules. Analog peptides can becreated by substituting amino acid residues at primary anchor, secondaryanchor, or at primary and secondary anchor positions. Generally, analogsare made for peptides that already bear a motif or supermotif. For anumber of the motifs or supermotifs in accordance with the invention,residues are defined which are deleterious to binding to allele-specificHLA molecules or members of HLA supertypes that bind the respectivemotif or supermotif (see, e.g., Rupert et al. Cell 74:929, 1993; Sidney,J. et al., Hu. Immunol. 45:79, 1996; and Sidney et al.; Sidney, et al.,J. Immunol. 154:247, 1995). Accordingly, removal of such residues thatare detrimental to binding can be performed in accordance with thepresent invention. For example, in the case of the A3 supertype, whenall peptides that have such deleterious residues are removed from thepopulation of peptides used in the analysis, the incidence ofcross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. etal., Hu. Immunol. 45:79, 1996).

Thus, one strategy to improve the cross-reactivity of peptides within agiven supermotif is simply to delete one or more of the deleteriousresidues present within a peptide and substitute a small “neutral”residue such as Ala (that may not influence T cell recognition of thepeptide). An enhanced likelihood of cross-reactivity is expected if,together with elimination of detrimental residues within a peptide,“preferred” residues associated with high affinity binding to anallele-specific HLA molecule or to multiple HLA molecules within asuperfamily are inserted.

In some embodiments, a T helper peptide can used in addition to one ofthe peptides of the invention. One type of T helper peptide is one thatis recognized by T helper cells in the majority of the population. Thiscan be accomplished by selecting amino acid sequences that bind to many,most, or all of the MHC class II molecules. These are known as “looselyMHC-restricted” T helper sequences. Examples of amino acid sequencesthat are loosely MHC-restricted include sequences from antigens such asTetanus toxin at positions 830-843 (QYIKANSKFIGITE (SEQ ID NO: 1)),Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398(DIEKKIAKMEKASSVFNVVNS (SEQ ID NO: 2)), and Streptococcus 18 kD proteinat positions 1-16 (YGAVDSILGGVATYGAA (SEQ ID NO: 3)).

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely MHC-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds, calledPan-DR-binding epitopes or PADRE® molecules (Epimmune, San Diego,Calif.), are designed on the basis of their binding activity to mostHLA-DR (human MHC class II) molecules (see, e.g., U.S. Ser. No.08/121,101 (now abandoned) and related U.S. Ser. No. 08/305,871 (nowU.S. Pat. No. 5,736,142)). For instance, a pan-DR-binding epitopepeptide having the formula: aKXVWANTLKAAa (SEQ ID NO: 4), where X iseither cyclohexylalanine, phenylalanine, or tyrosine, and “a” is eitherD-alanine or L-alanine, has been found to bind to most HLA-DR alleles,and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type.

Particularly preferred immunogenic peptides and/or T helper conjugatesare linked by a spacer molecule. The spacer is typically comprised ofrelatively small, neutral molecules, such as amino acids or amino acidmimetics, which are substantially uncharged under physiologicalconditions. The spacers are typically selected from, e.g., Ala, Gly, orother neutral spacers of nonpolar amino acids or neutral polar aminoacids. It will be understood that the optionally present spacer need notbe comprised of the same residues and thus may be a hetero- orhomo-oligomer. When present, the spacer will usually be at least one ortwo residues, more usually three to six residues. Alternatively, the HTLpeptide may be linked to the T helper peptide without a spacer.

The immunogenic peptide may be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of theHTL peptide. The amino terminus of either the immunogenic peptide or theT helper peptide may be acylated. The T helper peptides used in theinvention can be modified in the same manner as HTL peptides. Forinstance, they may be modified to include D-amino acids or be conjugatedto other molecules such as lipids, proteins, sugars and the like.Exemplary T helper peptides include tetanus toxoid 830-843, influenza307-319, malaria circumsporozoite 382-398 and 378-389.

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes HTLand CTL. Lipids have been identified as agents capable of priming HTLand CTL in vivo against viral antigens. For example, palmitic acidresidues can be attached to the alpha and epsilon amino groups of a Lysresidue and then linked, e.g., via one or more linking residues such asGly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be injected directly in a micellar form,incorporated into a liposome or emulsified in an adjuvant, e.g.,incomplete Freund's adjuvant. In a preferred embodiment a particularlyeffective immunogen comprises palmitic acid attached to alpha andepsilon amino groups of Lys, which is attached via linkage, e.g.,Ser-Ser, to the amino terminus of the immunogenic peptide. Also in apreferred embodiment a particularly effective immunogen comprisespalmitic acid attached to alpha and epsilon amino groups of Lys, whichis attached via linkage, e.g., Ser-Ser, to the amino terminus of a classI restricted peptide having T cell determinants, such as those peptidesdescribed herein as well as other peptides which have been identified ashaving such determinants

As another example of lipid priming of HTL and CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific HTL CTL when covalentlyattached to an appropriate peptide. See, Deres et al., Nature342:561-564 (1989), incorporated herein by reference. Peptides of theinvention can be coupled to P₃CSS, for example, and the lipopeptideadministered to an individual to specifically prime a HTL response tothe target antigen. Further, as the induction of neutralizing antibodiescan also be primed with P₃CSS conjugated to a peptide which displays anappropriate epitope, the two compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses toinfection.

In addition, additional amino acids can be added to the termini of apeptide to provide for ease of linking peptides one to another, forcoupling to a carrier support, or larger peptide, for modifying thephysical or chemical properties of the peptide or oligopeptide, or thelike. Amino acids such as tyrosine, cysteine, lysine, glutamic oraspartic acid, or the like, can be introduced at the C- or N-terminus ofthe peptide or oligopeptide. Modification at the C terminus in somecases may alter binding characteristics of the peptide. In addition, thepeptide or oligopeptide sequences can differ from the natural sequenceby being modified by terminal-NH2 acylation, e.g., by alkanoyl (C₁-C₂₀)or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia,methylamine, etc. In some instances these modifications may providesites for linking to a support or other molecule.

The peptides of the invention can be prepared in a wide variety of ways.Because of their relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co. (1984), supra.

Another aspect of the present invention is directed to vaccines whichcomprise an immunogenically effective amount of one or more peptides asdescribed herein. Peptides may be introduced into a host using a varietyof delivery vehicles known to those of skill in the art including PLGmicrospheres with entrapped peptides and virus-like particles.Furthermore, epitopes may be introduced as multiple antigen peptides(MAPs) (see e.g., Mora and Tam, J. Immunol. 161:3616-23 (1998)), or asimmunostimulating complexes (ISCOMS) (see e.g., Hu et al. Clin. Exp.Immunol. 113:235-43 (1998)) as known in the art.

Vaccines that contain an immunogenically effective amount of one or morepeptides as described herein are a further embodiment of the invention.The vaccines of the invention can be used both as a prevantative ortherapeutic. Once appropriately immunogenic epitopes have been defined,they can be delivered by various means, herein referred to as “vaccine”compositions. Such vaccine compositions can include, for example,lipopeptides (e.g., Vitiello, A. et al., J: Clin. Invest. 95:341, 1995),peptide compositions encapsulated in poly(DL-lactide-co-glycolide)(“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol.28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al.,Vaccine 13:675-681, 1995), peptide compositions contained in immunestimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-24: 1998),multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc.Nati. Acaa Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J Immunol. Methods196:17-32, 1996), vir delivery vectors (Perkus, M. E. et al., In:Concepts in vaccine development, Kaufmann H. E., ed., p. 379, 1996;Chakrabarti, S. et al., Nature 320:535,1986; Hu, S. L. et al., Nature320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986;Top, F. et al., J Infect. Dis. 124:148, 1971; Chanda, P. K. et al.,Virology 175:535, 1990), particles of viral or synthetic origin (e.g.,Kofler, N. et al., J Immunol. Methods. 192:2- 1996; Eldridge, J. H. etal., Sem. Bematol. 30:16, 1993; Fa10, L. D., Jr. et al., Nature Med.7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A.Annu. Re Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293,1993), liposomes (Reddy, R et al., J. Immunol. 148:1585, 1992; Rock, K.L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA(Ulmer, J. B. et al., Science 259:1745, 1993; Robinsol H. L., Hunt, L.A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In:Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996;Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 andEldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeteddelivery technologies, also known as receptor mediated targeting, suchas those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) mayalso be used.

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA encoding one or more of the peptides of theinvention can also be administered to a patient. This approach isdescribed, for instance, in Wolff et. al., Science 247: 1465 (1990) aswell as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566;

5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below.Examples of DNA-based delivery technologies include “naked DNA”,facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationiclipid complexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, the peptides ofthe invention can be expression vectors include attenuated viral hosts,such as vaccinia or fowlpox. This approach involves the use of vacciniavirus, for example, as a vector to express nucleotide sequences thatencode the pep tides of the invention. Upon introduction into an acutelyor chronically infected host or into a non-infected host, therecombinant vaccinia virus expresses the immunogenic peptide, andthereby elicits a host CTL and/or HTL response. Vaccinia vectors andmethods useful in immunization protocols are described in, e.g., U.S.Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCGvectors are described in Stover et al., Nature 351:456-460 (1991). Awide variety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g. adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent to those skilled in the art from the description herein.

Furthermore, vaccines in accordance with the invention can encompass oneor more of the peptides of the invention. Accordingly, a peptide can bepresent in a vaccine individually. Alternatively, the peptide can beindividually linked to its own carrier; alternatively, the peptide canexist as a homopolymer comprising multiple copies of the same peptide,or as a heteropolymer of various peptides. Polymers have the advantageof increased immunological reaction and, where different peptideepitopes are used to make up the polymer, the additional ability toinduce antibodies and/or CTLs that react with different antigenicdeterminants of the pathogenic organism or tumor-related peptidetargeted for an immune response. The composition may be a naturallyoccurring region of an antigen or may be prepared, e.g., recombinantlyor by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, CTL responses can be primed by conjugating peptidesof the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS).

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of HTLsand/or CTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later infection, or at leastpartially resistant to developing an ongoing chronic infection, orderives at least some therapeutic benefit when the antigen wastumor-associated.

For therapeutic or immunization purposes, the peptides of the inventioncan also be expressed by vectors. Examples of expression vectors includeattenuated viral hosts, such as vaccinia or fowlpox. This approachinvolves the use of vaccinia virus as a vector to express nucleotidesequences that encode the peptides of the invention. Vaccinia vectorsand methods useful in immunization protocols are described in, e.g.,U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille CalmetteGuerin). BCG vectors are described in Stover, et al. Nature 351:456-60(1991). A wide variety of other vectors useful for therapeuticadministration or immunization of the peptides of the invention, e.g.,Salmonella typhi vectors, retroviral vectors, adenoviral oradeno-associated viral vectors, and the like will be apparent to thoseskilled in the art from the description herein.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982) (also1989), which is incorporated herein by reference. Thus, fusion proteinswhich comprise one or more peptide sequences of the invention can beused to present the appropriate T cell epitope. For example, a codingsequence encoding a peptide of the invention can be provided withappropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein. A number of such vectors andsuitable host systems are now available. Expression constructs, i.e.,minigenes are described in greater detail in the sections below. Suchmethodologies are also used to present at least one peptide of theinvention along with a substance which is not a peptide of theinvention.

As the coding sequence for peptides of the length contemplated hereincan be synthesized by chemical techniques, for example, using thephosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185(1981), with modification can be made simply by substituting theappropriate base(s) for those encoding the native peptide sequence. Thecoding sequence can then be provided with appropriate linkers andligated into expression vectors commonly available in the art, and thevectors used to transform suitable hosts to produce the desired fusionprotein. A number of such vectors and suitable host systems are nowavailable. For expression of the fusion proteins, the coding sequencewill be provided with operably linked start and stop codons, promoterand terminator regions and usually a replication system to provide anexpression vector for expression in the desired cellular host. Forexample, promoter sequences compatible with bacterial hosts are providedin plasmids containing convenient restriction sites for insertion of thedesired coding sequence. The resulting expression vectors aretransformed into suitable bacterial hosts. Of course, yeast or mammaliancell hosts may also be used, employing suitable vectors and controlsequences.

The peptides of the present invention and pharmaceutical and vaccinecompositions thereof are useful for administration to mammals,particularly humans, to treat and/or prevent cancer.

In therapeutic applications, compositions are administered to a patientin an amount sufficient to elicit an effective HTL response to the tumorantigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose” or “unit dose.” Amounts effective forthis use will depend on, e.g., the peptide composition, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician, but generally range for the initialimmunization (that is for therapeutic or prophylactic administration)from about 1.0 μg to about 5000 μg of peptide for a 70 kg patient,followed by boosting dosages of from about 1.0 μg to about 1000 μg ofpeptide pursuant to a boosting regimen over weeks to months dependingupon the patient's response and condition by measuring specific CTLactivity in the patient's blood. In alternative embodiments, generallyfor humans the dose range for the initial immunization (that is fortherapeutic or prophylactic administration) is from about 1.0 μg toabout 20,000 μg of peptide for a 70 kg patient, preferably, 100 μg-, 150μg-, 200 μg-, 250 μg-, 300 μg-, 400 μg-, or 500 μg-20,000 μg, followedby boosting dosages in the same dose range pursuant to a boostingregimen over weeks to months depending upon the patient's response andcondition by measuring specific HTL activity in the patient's blood. Inembodiments where recombinant nucleic acid administration is used, theadministered material is titrated to achieve the appropriate therapeuticresponse.

It must be kept in mind that the peptides and compositions of thepresent invention may generally be employed in serious disease states,that is, life-threatening or potentially life threatening situations. Insuch cases, in view of the minimization of extraneous substances and therelative nontoxic nature of the peptides, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these peptide compositions.

For therapeutic use, administration should begin at the first sign oftumors or shortly after diagnosis. This is followed by boosting dosesuntil at least symptoms are substantially abated and for a periodthereafter.

Treatment of an affected individual with the compositions of theinvention may hasten resolution of the infection in acutely infectedindividuals. For those individuals susceptible (or predisposed) todeveloping cancer the compositions are particularly useful in methodsfor preventing the evolution of cancer.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., water, buffered water, 0.8% saline,0.3% glycine, hyaluronic acid and the like. These compositions may besterilized by conventional, well known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

A pharmaceutical composition of the invention may comprise one or more Tcell stimulatory peptides of the invention. For example, apharmaceutical composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more T cell stimulatory peptides of the invention. Moreover, apharmaceutical composition of the invention may comprise one or more Tcell stimulatory peptides of the invention in combination with one ormore other T cell stimulatory peptides. The concentration of each uniqueT cell stimulatory peptide of the invention in the pharmaceuticalformulations can vary widely, e.g., from less than about 0.001%, about0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, 0.007%,0.008%, 0.009%, about 0.01%, about 0.02%, about 0.025%, about 0.03%,about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about1.8%, about 1.9%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 20%, to about 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.In a preferred embodiment, the concentration of each unique T cellstimulatory peptide of the invention in the pharmaceutical formulationsis about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%,about 0.006%, 0.007%, 0.008%, 0.009%, about 0.01%, about 0.02%, about0.025%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%,about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about1% by weight. In a more preferred embodiment, the concentration of eachunique T cell stimulatory peptide of the invention in the pharmaceuticalformulations is about 0.01%, about 0.02%, about 0.025%, about 0.03%,about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about0.09%, about 0.1% by weight.

The concentration of HTL stimulatory peptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.A human unit dose form of the peptide composition is typically includedin a pharmaceutical composition that comprises a human unit dose of anacceptable carrier, preferably an aqueous carrier, and is administeredin a volume of fluid that is known by those of skill in the art to beused for administration of such compositions to humans.

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue, or targeted selectively to infected cells, as well asincrease the half-life of the peptide composition.

Liposomes include emulsions, foams, micelles, insoluble monolayers,liquid crystals, phospholipid dispersions, lamellar layers and the like.In these preparations the peptide to be delivered is incorporated aspart of a liposome, alone or in conjunction with a molecule which bindsto, e.g., a receptor prevalent among lymphoid cells, such as monoclonalantibodies which bind to the CD45 antigen, or with other therapeutic orimmunogenic compositions. Thus, liposomes either filled or decoratedwith a desired peptide of the invention can be directed to the site oflymphoid cells, where the liposomes then deliver the selectedtherapeutic/immunogenic peptide compositions. Liposomes for use in theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028, and 5,019,369, incorporated herein by reference.

For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension containing a peptide may be administered intravenously,locally, topically, etc. in a dose which varies according to, interalia, the manner of administration, the peptide being delivered, and thestage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included, asdesired, as with, e.g., lecithin for intranasal delivery.

In another aspect the present invention is directed to vaccines whichcontain as an active ingredient an immunogenically effective amount ofan immunogenic peptide as described herein. The peptide(s) may beintroduced into a host, including humans, linked to its own carrier oras a homopolymer or heteropolymer of active peptide units. Such apolymer has the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies and/or CTLs that react with differentantigenic determinants of tumor cells. Useful carriers are well known inthe art, and include, e.g., thyroglobulin, albumins such as human serumalbumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamicacid), influenza, hepatitis B virus core protein, hepatitis B virusrecombinant vaccine and the like. The vaccines can also contain aphysiologically tolerable (acceptable) diluent such as water, phosphatebuffered saline, or saline, and further typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are materials well known in the art. Uponimmunization with a peptide composition as described herein, viainjection, aerosol, oral, transdermal or other route, the immune systemof the host responds to the vaccine by producing large amounts of HTLsspecific for the desired antigen, and the host becomes at leastpartially immune to later infection, or resistant to developing chronicinfection.

The peptides of the present invention and pharmaceutical and vaccinecompositions of the invention are useful for administration to mammals,particularly humans, to treat and/or prevent cancer. Vaccinecompositions containing the peptides of the invention are administeredto a patient susceptible to or otherwise at risk of cancer to elicit animmune response against the antigen and thus enhance the patient's ownimmune response capabilities. Such an amount is defined to be an“immunogenically effective dose.” In this use, the precise amounts againdepend on the patient's state of health and weight, the mode ofadministration, the nature of the formulation, etc., but generally rangefrom about 1.0 μg to about 5000 μg per 70 kilogram patient, morecommonly from about 10 μg to about 500 μg mg per 70 kg of body weight.

As noted herein, the peptides of the invention induce HTL immuneresponses when contacted with a HTL specific to an epitope comprised bythe peptide. The manner in which the peptide is contacted with the HTLis not critical to the invention. For instance, the peptide can becontacted with the HTL either in vivo or in vitro. If the contactingoccurs in vivo, the peptide itself can be administered to the patient orother vehicles, e.g., DNA vectors encoding one or more peptide, viralvectors encoding the peptide(s), liposomes and the like, can be used, asdescribed herein.

For therapeutic or immunization purposes, nucleic acids encoding one ormore of the peptides of the invention can also be administered to thepatient. A number of methods are conveniently used to deliver thenucleic acids to the patient. For instance, the nucleic acid can bedelivered directly, as “naked DNA”. This approach is described, forinstance, in Wolff et. al., Science 247: 1465-68 (1990) as well as U.S.Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also beadministered using ballistic delivery as described, for instance, inU.S. Pat. No. 5,204,253. Particles comprised solely of DNA can beadministered. Alternatively, DNA can be adhered to particles, such asgold particles.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in WO 96/18372; WO 93/24640; Mannino andGould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No.5,279,833; WO 91/06309; and Feigner et a/. (1987) Proc. Natl. Acad. Sci.USA 84: 7413-14.

Nucleic acids encoding one or more of the peptides of the invention canalso be administered to the patient. This approach is described, forinstance, in Wolff, et. al., Science, 247:1465-68 (1990) as well as U.S.Pat. Nos. 5,580,859 and 5,589,466.

A preferred means of administering nucleic acids encoding the peptidesof the invention uses minigene constructs encoding multiple epitopes ofthe invention. To create a DNA sequence encoding the selected HTLepitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes are reverse translated. A human codon usagetable is used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences are directly adjoined, creating acontinuous polypeptide sequence. To optimize expression and/orimmunogenicity, additional elements can be incorporated into theminigene design. Examples of amino acid sequence that could be reversetranslated and included in the minigene sequence include: a leader(signal) sequence, and an endoplasmic reticulum retention signal. Inaddition, MHC presentation of HTL epitopes may be improved by includingsynthetic (e.g. poly-alanine) or naturally-occurring flanking sequencesadjacent to the HTL epitopes.

The minigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the HTL epitope polypeptide,can then cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the artare included in the vector to ensure expression in the target cells.Several vector elements are required: a promoter with a down-streamcloning site for minigene insertion; a polyadenylation signal forefficient transcription termination; an E. coli origin of replication;and an E. coli selectable marker (e.g. ampicillin or kanamycinresistance). Numerous promoters can be used for this purpose, e.g., thehuman cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencescan also be considered for increasing minigene expression. It hasrecently been proposed that immunostimulatory sequences (ISSs or CpGs)play a role in the immunogenicity of DNA vaccines. These sequences couldbe included in the vector, outside the minigene coding sequence, iffound to enhance immunogenicity.

In some embodiments, a bicistronic expression vector, to allowproduction of the minigene-encoded epitopes and a second proteinincluded to enhance or decrease immunogenicity can be used. Examples ofproteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF),cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules.Helper (HTL) epitopes could be joined to intracellular targeting signalsand expressed separately from the CTL epitopes. This would allowdirection of the HTL epitopes to a cell compartment different than theCTL epitopes. If required, this could facilitate more efficient entry ofHTL epitopes into the MHC class II pathway, thereby improving CTLinduction. In contrast to CTL induction, specifically decreasing theimmune response by co-expression of immunosuppressive molecules (e.g.TGF-β) may be beneficial in certain diseases.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coil strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

Therapeutic quantities of plasmid DNA are produced by fermentation in E.coli, followed by purification. Aliquots from the working cell bank areused to inoculate fermentation medium (such as Terrific Broth), andgrown to saturation in shaker flasks or a bioreactor according to wellknown techniques. Plasmid DNA can be purified using standardbioseparation technologies such as solid phase anion-exchange resinssupplied by Quiagen. If required, supercoiled DNA can be isolated fromthe open circular and linear forms using gel electrophoresis or othermethods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques may become available. As noted above,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

The nucleic acids can also be administered using ballistic delivery asdescribed, for instance, in U.S. Pat. No. 5,204,253. Particles comprisedsolely of DNA can be administered. Alternatively, DNA can be adhered toparticles, such as gold particles.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanMHC molecules are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g. IM for DNA in PBS, IP forlipid-complexed DNA). Twenty-one days after immunization, splenocytesare harvested and restimulated for 1 week in the presence of peptidesencoding each epitope being tested.

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes in HLA molecules on their surfaces.

Dendritic cells can also be transfected, e.g., with a minigenecomprising nucleic acid sequences encoding the epitopes in accordancewith the invention, in order to elicit immune responses. Vaccinecompositions can be created in vitro, following dendritic cellmobilization and harvesting, whereby loading of dendritic cells occursin vitro.

Transgenic animals of appropriate haplotypes may additionally provide auseful tool in optimizing the in vivo immunogenicity of minigene DNA. Inaddition, animals such as monkeys having conserved HLA molecules withcross reactivity to CTL epitopes recognized by human MHC molecules canbe used to determine human immunogenicity of CTL epitopes (Bertoni, etal., J. Immunol. 161:4447-4455 (1998)).

Such in vivo studies are required to address the variables crucial forvaccine development, which are not easily evaluated by in vitro assays,such as route of administration, vaccine formulation, tissuebiodistribution, and involvement of primary and secondary lymphoidorgans. Because of their simplicity and flexibility, HLA transgenic micerepresent an attractive alternative, at least for initial vaccinedevelopment studies, compared to more cumbersome and expensive studiesin higher animal species, such as nonhuman primates.

Antigenic peptides are used to elicit a HTL response ex vivo, as well.The resulting HTL cells, can be used to treat tumors in patients that donot respond to other conventional forms of therapy, or will not respondto a therapeutic vaccine peptide or nucleic acid in accordance with theinvention. Ex vivo HTL responses to a particular antigen are induced byincubating in tissue culture the patient's (HTLp), or geneticallycompatible, HTL precursor cells together with a source ofantigen-presenting cells (APC), such as dendritic cells, and theappropriate immunogenic peptide. After an appropriate incubation time(typically about 7-28 days (1-4 weeks)), in which the precursor cellsare activated and matured and expanded into effector cells, the cellsare infused back into the patient, where they will destroy theirspecific target cell (an infected cell or a tumor cell). Transfecteddendritic cells may also be used as antigen presenting cells. In orderto optimize the in vitro conditions for the generation of specifichelper T cells, the culture of stimulator cells is maintained in anappropriate serum-free medium.

The peptides may also find use as diagnostic reagents. For example, apeptide of the invention may be used to determine the susceptibility ofa particular individual to a treatment regimen which employs the peptideor related peptides, and thus may be helpful in modifying an existingtreatment protocol or in determining a prognosis for an affectedindividual. In addition, the peptides may also be used to predict whichindividuals will be at substantial risk for developing chronicinfection.

For example, a peptide of the invention may be used in a tetramerstaining assay to assess peripheral blood mononuclear cells for thepresence of antigen-specific CTLs following exposure to a pathogen orimmunogen. The HLA-tetrameric complex is used to directly visualizeantigen-specific CTLs (see, e.g., Ogg, et al. Science 279:2103-2106,1998; and Altman, et al. Science 174:94-96, 1996) and determine thefrequency of the antigen-specific CTL population in a sample ofperipheral blood mononuclear cells. A tetramer reagent using a peptideof the invention may be generated as follows: A peptide that binds to anallele-specific HLA molecule or supertype molecule is refolded in thepresence of the corresponding HLA heavy chain and 132-microglobulin togenerate a trimolecular complex. The complex is biotinylated at thecarboxyl terminal end of the heavy chain at a site that was previouslyengineered into the protein. Tetramer formation is then induced by theaddition of streptavidin. By means of fluorescently labeledstreptavidin, the tetramer can be used to stain antigen-specific cells.The cells may then be identified, for example, by flow cytometry. Suchan analysis may be used for diagnostic or prognostic purposes. Inaddition, the peptides may also be used to predict which individualswill be at substantial risk for developing chronic infection.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent or patent application were specifically andindividually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modificationsmay be made thereto without departing from the spirit or scope of theappended claims.

EXAMPLES Materials and Methods

The following materials and methods apply generally to all the examplesdisclosed herein. Specific materials and methods are disclosed in eachexample, as necessary.

Reagents: Anti-IFN-γ and biotinylated anti-IFN-γ were obtained fromMabtech (Sweden). Phorbol myristate acetate (PMA), human serum albumin(HSA), polyclonal human IgG, tetanus toxin (TT), and ionomycin were fromSigma (St. Louis, Mo., USA). Goat anti-human horseradish peroxidase(HRP)-conjugated antibody was obtained from Santa Cruz Biotechnology(Santa Cruz, Calif.). Hank's balanced salts solution (HBSS), RPMI-1640and phosphate-buffered saline were from Cellgro (Hernden, Va., USA).Ficoll-Paque was from Amersham Biosciences (Uppsala, Sweden). Allpeptides were synthesized by either the Mayo Clinic Protein Chemistryand Proteomics Core or by Epimmune, Inc. (San Diego, Calif.) andpurified to >95% homogeneity by reverse-phase HPLC as previouslydescribed (Dzuris J L, Sidney J, Appella E, Chesnut R W, Watkins D I,Sette A. Conserved MHC class I peptide binding motif between humans andrhesus macaques. J Immunol 2000;164: 283-91). Purity of peptides wasdetermined with reverse-phase HPLC and amino acid analysis, sequencing,and/or mass spectrometry. Lyophilized peptides were resuspended at 20mg/ml in 100% DMSO and then diluted to required concentrations in PBS.

Epitope Prediction: The prediction program used, PIC (Predicted IC50),is a modified linear coefficient, or matrix-based method for predictingpeptides with HLA-DR binding capacity. PIC is predicated on theassumption that each residue along a peptide molecule can independentlycontribute to binding affinity (Sette A, et al. Proc Natl Acad Sci U S A1989;86: 3296-300; Sette A, et al. J Immunol 1989;142: 35-40). Thealgorithm yields a predicted IC50 value (designated as PIC) for thecorresponding input sequence. Lower PIC values indicate a higherprobability of binding to HLA. The program analyzes 15 amino acid longsequences offset by 3 residues encompassing the entire protein.

Peripheral Blood Mononuclear Cell Preparation (PBMC): PBMC were isolatedfrom blood as described (Disis M L, et al. Clin Cancer Res 1999;5:1289-97), and cryopreserved in liquid nitrogen (20×106/ml cells) infreezing media (RPMI with 12.5% HSA, penicillin, streptomycin and 2 mMglutamine) (Disis ML et al. J Immunol Methods 2005.).

HLA-DR purification. Fifteen distinct HLA-DR molecules were used inquantitative assays to measure the binding of peptides to solubilizedHLA-DR molecules. These HLA-DR molecules were chosen to allow balancedpopulation coverage: DRB1*0101, DRB1*1501, DRB1*0301, DRB1*0401,DRB1*0404, DRB1*1101, DRBS*0101, DRB4*0101, DRB3*0101, DRB1*0701,DRB1*0405, DRB1*0802, DRB1*0901, DRB1*1201, and DRB1*1302 (24). MHCmolecules utilized were purified from EBV transformed homozygous celllines or single MHC allele transfected 721.221, C1R, or fibroblastlines. The cell lines were maintained by culture in RPMI-1640 mediumsupplemented with 2 mM L-glutamine, 100 U (100 μg/ml)penicillin-streptomycin solution, and 10% heat-inactivated FCS. HLA-DRmolecules were purified using antibody-based affinity chromatographyfrom cell lysates prepared in 50 mM Tris-HCL, pH 8.5, containing 1%(v/v) NP-40, 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. Briefly, columns ofinactivated Sepharose CL4B and Protein A Sepharose were used aspre-columns. HLA-DR molecules were captured by passage of lysates overLB3.1 monoclonal antibody (anti-HLA-DRA) columns. Antibody columns werewashed with 10 mM Tris-HCL, pH8.0 with 1% (v/v) NP-40, followed by PBScontaining 0.4% (w/v) n-octylglucoside. MHC molecules were then elutedwith 50 mM diethylamine in 0.15 M NaCl containing 0.4% (w/v)n-octylglucoside, pH 11.5. The pH was reduced to 8.0 and the eluateswere concentrated by centrifugation in Centriprep 30 concentrators at2000 rpm (Amicon, Beverly, Mass.).

HLA-DR binding assays: Radioligand binding inhibition assays were usedto measure the binding of peptides to soluble HLA-DR molecules based onthe inhibition of binding of a radiolabeled standard peptide asdescribed previously (Sidney J, Southwood S, Oseroff C, del Guercio M F,Grey H M, Sette A. Measurement of MHC/peptide interactions by gelfiltration. Curr Protocols Immunol 1998;18: 18.3.2-.3.9.). Briefly, 1-10nM of radiolabeled peptide was co-incubated for 2 days at either roomtemperature or 37° C. with 1 μM to 1 nM purified HLA-DR molecules in thepresence of a cocktail of protease inhibitors. Assays were performed atvarious pH conditions, ranging from pH 4 to pH 7. The final pH of assaymixtures is adjusted using citrate buffer as described elsewhere (SidneyJ, Curr Protocols Immunol 1998). After incubation, the percentage ofHLA-DR-bound radioactivity is determined by capturing HLA-DR/peptidecomplexes on Optiplates (Packard Instruments, Meriden, Conn.) coatedwith the LB3.1 antibody and determining bound counts per minute usingthe TopCount microscintillation counter (Packard Instruments). Theamount of HLA-DR yielding 10-20% bound radioactivity is used in theinhibition assays in which the concentration of peptide yielding 50%inhibition of the binding of the radiolabeled peptide was calculated.Under the conditions used, the measured IC₅₀ values are reasonableapproximations of the true Kd values. Competitor peptides are tested in2-4 complete, independent experiments, at concentrations ranging from 30μg/mL to 300 μg/mL. As in previous studies, peptides with affinities forspecific HLA-DR molecules of 1000 nM or better are defined as bindersfor the respective antigens.

Enzyme-linked immunosorbent spot assay. A 10-day ELlspot for detectinglow-frequency T cells was used to determine reactivity to the tumorantigen peptides (Table 1) as described (Knutson K L et al. J Clin Onc2006;24: 4254-61). A positive response to a peptide was defined as afrequency that was significantly (p<0.05, two-tailed t test) greaterthan the mean of control no-antigen wells and detectable(i.e., >1:100,000). PMA/Ionomycin and the CEF pool were used as positivenon-tumor related controls as previously described (Knutson, 2006).

ELISA. ELISAs were done as previously described (Knutson, 2006).Briefly, 96-well plates were coated with 1 μg/ml IGFBP-2 protein, 200ng/ml tetanus toxin or 1 μg/ml BSA. Human IgG was added at aconcentration range of 200 to 0.2 ng/ml to some wells for standard curvegeneration. After washing and blocking, human sera were added to theplate at a 1:40 dilution in triplicate and plates were incubated for 2hr at RT. After washing, 100 μL/well of HRP (Santa Cruz Biotechnology)was diluted 1:2000 and incubated for 1 hr at RT. After a final wash,each well was incubated with 100 μL (tetramethylbenzadine) TMB substrate(BD Bioscience). Color development was stopped with diluted HCL andabsorbance was read at 450 nm on a plate reader.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

Example 1

Identification of Conserved HLA Class II- Restricted Peptides Derivedfrom Tumor Associated Antigens Using Established Motif Search Algorithms

To identify epitopes useful for vaccine design, a multidisciplinaryapproach was used based initially on amino acid motif searching of tumorassociated antigen sequences to identify potential HLA Class II motifs(see Table I). This was followed by high throughput synthetic peptidebinding assays using purified HLA molecules to determine affinity andbreadth of epitope peptide binding.

Algorithm motif searches: Motif search algorithms were validated for themost common HLA Class II alleles and were focused on the HLA DRB1*0101,DRB1*1501, DRB1*0301, DRB1*0401, DRB1*0404, DRB1*1101, DRB5*0101,DRB4*0101, DRB3*0101, DRB1*0701, DRB1*0405, DRB1*0802, DRB1*0901,DRB1*1201, and DRB1*1302 supertypes in order to attain virtually 100%population coverage. The selected tumor associated antigen sequenceswere scanned for motif positive amino acid sequences using the motifdefinitions.

A total of about 150 Class II-restricted peptide sequences wereidentified that were specific for various DR supertypes (see Table I).Table I lists for each identified DR antigen peptide, the IC₅₀ (nM) foreach purified HLA.

Example 2

Identification of HTL Epitopes for Tumor Vaccine Inclusion Of thepeptides listed in Table I, those peptides that bound to at least 4different HLA with an IC₅₀ of less than 1000 nM were identified and areshown in Table II. The peptide sequences of Table II were furtherevaluated for their binding capacity to purified MHC molecules. FIGS. 1through 4 show those peptides of Table II which were immunogenic (shownin a circle). These HTL peptides are candidates for inclusion into atumor vaccine.

Using predictive algorithms discussed above, candidate HLA-DR1-bindingepitopes were identified. Binding assays targeting 15 different HLA-DRmolecules revealed that 10 of the epitopes were indiscriminate, binding(IC₅₀<1000 nM) to at least four different HLA-DR variants. Aninterferon-gamma ELIspot assay was used to assess immunity to theindiscriminate binding peptides in 48 patients with either breast orovarian cancer and 18 healthy controls. The results showed that elevatedT cell immunity in patients was detected to several peptides (FIGS.1-4).

Healthy donor and patient samples were obtained. Patients were free fromactive treatment for at least 30 days when blood (200 ml) was collected.For the T cell studies, the mean (±s.e.m) ages of the healthy donors andpatients were 42±11 and 55±2 years, respectively (p<0.0001). Due to seraunavailability, not all of the controls used in the T cell studies wereexamined for tumor associated antigen antibodies in their sera. However,additional control and patient sera were available for antibodyassessment. Additional healthy donor sera was obtained fromBioreclamation (Hicksville, N.Y.).

Using an ELlspot assay with a limit of detection of approximately1:100,000 antigen-specific T cells per million PBMC, patient-derivedPBMC were screened for reactivity against all of the HLA-DR bindingpeptides. The immunogenicity data of several peptides is shown in FIGS.1-4.

Example 3 Design and Development of Multi-Epitope Vaccines

Peptides that bound to at least 4 different HLA subtypes as describedabove, and were shown to be immunogenic, as shown in FIGS. 1-4, wereselected for inclusion in a multi-epitope vaccine. Table III shows thepercent of patients that demonstrated positive responses and the bindingpatterns to specific HLA subtypes of eight vaccine candidates.

These example and equivalents thereof will become more apparent to thoseskilled in the art in light of the present disclosure and theaccompanying claims. It should be understood, however, that the examplesare designed for the purpose of illustration only and not limiting ofthe scope of the invention in any way.

TABLE I HLA-DR binding affinity of Breast/Ovarian-derived peptidesIC₅₀ nM purified HLA DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 Peptide SequenceSource Protein Position *0101 *0301 *0401 *0404 *0405 *0701 9019.0104RWCIPWQRLLLTASL CEA.10 CEA 10 1467 7860 426 193 221 107   9019.0105LLTFWNPPTTAKLTI CEA.24 CEA 24 6.9 16,313 273 52 258 3.7 9019.0106TAKLTIESTPFNVAE CEA.33 CEA 33 72 613 106 41 383 70 9019.0107EVLLLVHNLPQHLFG CEA.50 CEA 50 2.7 830 3.4 1.7 30 5.4 9019.0108YSWYKGERVDGNRQI CEA.65 CEA 65 511 — 34 585 360 866 9019.0109NRQIIGYVIGTQQAT CEA.76 CEA 76 216 — 108 1.5 129 46 9019.0110QYVIGTQQATPGPAY CEA.81 CEA 81 21 31 34 4210 1705 9019.0111GPAYSGREIIYPNAS CEA.92 CEA 92 9435 12,989 5252 9019.0112 GREIIYPNASLLIQNCEA.97 CEA 97 62 433 251 88 550 29 9019.0113 DIGFYTLHVIKSDLV CEA.116 CEA116 64 984 84 260 95 23 9019.0114 FLTLHVIKSDLVNEE CEA.119 CEA 119 101 80184 169 41 56 9019.0005 LHVIKSDLVNEEATG CEA.122 CEA 122 891 46 214 10337 3893 9019.0115 KSDLVNEEATGQFRV CEA.126 CEA 126 13,600 2530 236 63389019.0116 QFRVYPELPKPSISS CEA.137 CEA 137 1780 1727 2916 7976 9019.0117KPSISSNNSKPVEDK CEA.146 CEA 146 405 919 2111 6959 407 9019.0118YLWWVNNQSLPVSPR CEA.176 CEA 176 2.4 100 832 203 80 17 9019.0119SDSVILNVLYGPDAP CEA.226 CEA 226 111 255 314 1453 5236 9019.0120LNVLYGPDAPTISPL CEA.231 CEA 231 331 649 1378 — 410 9019.0121APTISPLNTSYRSGE CEA.239 CEA 239 2431 295 49 5994 10,607 9019.0122QYSWFVNGTFQQSTQ CEA.268 CEA 268 5983 21 7830 364 216 9019.0123QELFIPNITVNNSGS CEA.282 CEA 282 147 644 25 227 379 1658 9019.0124RTTVTTITVYAEPPK CEA.310 CEA 310 4115 259 697 32 649 9019.0125TITVYAEPPKPFITS CEA.315 CEA 315 12,755 539 — 12,658 5704 8230 9019.0126YLWWVNNQSLPVSPR CEA.354 CEA 354 1123 234 12 248 88 28 9019.0127SDPVILNVLYGPDDP CEA.404 CEA 404 384 592 347 3732 2248 9019.0128SYTYYRPGVNLSLSC CEA.423 CEA 423 1.6 4425 6.8 4036 300 5.4 9019.0129YSWLIDGNIQQHTQE CEA.447 CEA 447 1407 95 9827 9019.0130 NSGLYTCQANNSASGCEA.471 CEA 471 49 33 37 96 8139 9019.0131 RTTVKTITVSAELPK CEA.488 CEA488 89 1267 58 54 11 4.2 9019.0132 TITVSAELPKPSISS CEA.493 CEA 493 22374 9393 6997 3624 29 9019.0133 KPSISSNNSKPVEDK CEA.502 CEA 502 146 4031404 5564 121 9019.0134 YLWWVNGQSLPVSPR CEA.532 CEA 532 1.4 2.5 1356 18812 9019.0135 VCGIQNSVSANRSDP CEA.570 CEA 570 44 72 26 1854 10959019.0136 QNSVSANRSDPVTLD CEA.574 CEA 574 240 511 1432 — 274 9019.0137SSYLSGANLNLSCHS CEA.603 CEA 603 4.0 580 1596 7822 465 9019.0138QYSWRINGIPQQHTQ CEA.624 CEA 624 472 43 1203 311 11,900 9019.0139INGIPQQHTQVLFIA CEA.629 CEA 629 682 181 680 942 31 9019.0140NGTYACFVSNLATGR CEA.650 CEA 650 839 818 11 558 30 20 9019.0141YACFVSNLATGRNNS CEA.653 CEA 653 183 774 225 41 327 531 9019.0142NNSIVKSITVSASGT CEA.665 CEA 665 34 103 43 1.8 128 34 9019.0143SITVSASGTSPGLSA CEA.671 CEA 671 2807 75 11 3374 1559 9019.0144SPGLSAGATVGIMIG CEA.680 CEA 680 3507 4191 2000 2380 92 1622.05TVGIMIGVLVGVALI CEA.688 CEA 688 384 — — — 4454 9019.0006 HQLLCCEVETIRRAYCyclin D1.3 Cyclin D1 3 953 21 746 256 4200 11,553 9019.0146DANLLNDRVLRAMLK Cyclin D1.19 Cyclin D1 19 1463 2342 — 9019.0147NDRVLRAMLKAEETC Cyclin D1.24 Cyclin D1 24 1178 1963 12,160 9019.0148RAMLKAEETCAPSVS Cyclin D1.29 Cyclin D1 29 407 — 360 375 12,517 77939019.0149 FKCVQKEVLPSMRKI Cyclin D1.45 Cyclin D1 45 4099 3615 16,0569019.0012 QKEVLPSMRKIVATW Cyclin D1.49 Cyclin D1 49 9825 111 12,182 11023720 481 9019.0150 LPSMRKIVATWMLEV Cyclin D1.53 Cyclin D1 53 8.5 826 23828 123 4.6 9019.0151 MRKIVATWMLWVCEE Cyclin D1.56 Cyclin D1 56 764 7953— 1982 249 9019.0011 MLEVCEEQKCEEEVF Cyclin D1.64 Cyclin D1 64 —9019.0152 EEEVFPLAMNYLDRF Cyclin D1.74 Cyclin D1 74 850 2247 3070 22281597 9019.0153 VFPLAMNYLDRFLSL Cyclin D1.77 Cyclin D1 77 146 107 23321567 609 3332 9019.0007 DRFLSLEPVKKSRLQ Cyclin D1.86 Cyclin D1 86 16 29018 61 159 1057 9019.0154 DRFLSLEPVKKSRLQ Cyclin D1.86 Cyclin D1 86 15 6059 251 1831 9019.0155 LEPVKKSRLQLLGAT Cyclin D1.91 Cyclin D1 91 102 5112695 13,659 2152 9019.0156 RLQLLGATCMFVASK Cyclin D1.98 Cyclin D1 98 12 —91 633 1439 468 9019.0157 TCMFVASKMKETIPL Cyclin D1.105 Cyclin D1 105262 342 5454 1744 17 9019.0158 ASKMKETIPLTAEKL Cyclin D1.110 Cyclin D1110 130 — 1992 — 220 1622.01 TIPLTAEKLCIYTDN Cyclin D1.116 Cyclin D1 116253 11,708 1230 — — — 9019.0159 KLCIYTDNSIRPEEL Cyclin D1.123 Cyclin D1123 1915 64 95 241 3172 1105 9019.0009 DNSIRPEELLQMELL Cyclin D1.129Cyclin D1 129 4554 147 4677 2803 16,500 6648 9019.0160 DNSIRPEELLQMELLCyclin D1.129 Cyclin D1 129 1533 1345 3440 9019.0161 PEELLQMELLLVNKLCyclin D1.134 Cyclin D1 134 1274 1027 313 603 401 9019.0010EELLQMELLLVNKLK Cyclin D1.135 Cyclin D1 135 3385 1622.06 LLQMELLLVNKLKWNCyclin D1.139 Cyclin D1 139 39 — 3539 6.1 387 2196 9019.0163MELLLVNKLKWNLAA Cyclin D1.140 Cyclin D1 140 46 9006 177 491 2411 17979019.0164 VNKLKWNLAAMTPHD Cyclin D1.145 Cyclin D1 145 46 1419 88 781 747382 9019.0165 KWNLAAMTPHDFIEH Cyclin D1.149 Cyclin D1 149 33 6249 3405653 122 1101 9019.0013 WNLAAMTPHDFIEHF Cyclin D1.150 Cyclin D1 150 64259019.0166 PHDFIEHFLSKMPEA Cyclin D1.157 Cyclin D1 157 1246 1299 11609019.0167 NKQIIRKHAQTFVAL Cyclin D1.174 Cyclin D1 174 4.7 561 6.5 23 254.4 9019.0168 AQTFVALCATDVKFI Cyclin D1.182 Cyclin D1 182 8.4 551 26 13355 13 9019.0169 VKFISNPPSMVAAGS Cyclin D1.193 Cyclin D1 193 6.7 4128 1218 117 304 9019.0170 PPSMVAAGSVVAAVQ Cyclin D1.199 Cyclin D1 199 6.8 —12 26 1718 133 9019.0171 VAAVQGLNLRSPNNF Cyclin D1.209 Cyclin D1 209 4418,558 226 532 4248 161 9019.0172 IEALLESSLRQAQQN Cyclin D1.251Cyclin D1 251 2608 18 — 9019.0014 EALLESSLRQAQQNM Cyclin D1.252Cyclin D1 252 8212 151 605 1019 12,908 — 9019.0024 LAALCRWGLLLALLPHer2/neu.3 Her2/neu 3 2044 5394 7995 9019.0025 WGLLLALLPPGAASTHer2/neu.9 Her2/neu 9 1687 750 0.93 219 16,366 1622.02 LLALLPPGAASTQVCHer2/neu.12 Her2/neu 12 17 5146 118 — 7181 9019.0027 GTDMKLBLPASPETHHer2/neu.28 Her2/neu 28 2117 1693 6814 9019.0028 RHLYQGCQVVQGNLEHer2/neu.47 Her2/neu 47 1435 422 4948 9019.0029 GCQVVQGNLELTYLPHer2/neu.52 Her2/neu 52 2600 2739 1972 9019.0030 NLELTYLPTNASLSFHer2/neu.59 Her2/neu 59 4.9 7356 6.2 2.7 38 7.2 9019.0031LTYLPTNASLSFLQD Her2/neu.62 Her2/neu 62 9.7 3364 19 16 80 15 9019.0032IQEVQGYVLIAHNQV Her2/neu.77 Her2/neu 77 57 7763 111 178 102 35 9019.0033VVLLAHNQVRQVPLQ Her2/neu.83 Her2/neu 83 28 454 93 104 1185 92 9019.0034HNQVRQVFLQRLRIV Her2/neu.88 Her2/neu 88 950 971 840 78 1303 80 9019.0035VRQVPLQRLRIVRGT Her2/neu.91 Her2/neu 91 3065 1796 218 9019.0001GTQLFEDNYALAVLD Her2/neu.104 Her2/neu 104 210 29 640 3923 14,921 1299019.0036 GDPLNNTTFVTGASP Her2/neu.120 Her2/neu 120 6356 7468 13,1729019.0037 TTPVTGASPGGLREL Her2/neu.126 Her2/neu 126 992 2417 675 13,1981843 9019.0038

Her2/neu.141 Her2/neu 141 712

110 1541 9019.0039

Her2/neu.146 Her2/neu 146 71 40 12 769 2486 9019.0040 GGVIIQRNPQLCYQDHer2/neu.151 Her2/neu 151 142 158 93 1845 14,279 9019.0041NPQLCYQDTILWNDI Her2/neu.158 Her2/neu 158 3653 369 — 9019.0042

Her2/neu.265 Her2/neu 265 101

136 1627 1324 9019.0043 STDVGSCTLVCPLHN Her2/neu.305 Her2/neu 305 2872265 — 2139 9019.0044 CYGLGMEHLREVRAV Her2/neu.342 Her2/neu 342 139 29701027 493 5122 271 9019.0045 MEHLREVRAVISANI Her2/neu.347 Her2/neu 3479.6 3913 513 12 200 9.7 9019.0046 LREVRAVTSANIQEF Her2/neu.350 Her2/neu350 17 43 8.2 50 12 9019.0047

Her2/neu.367 Her2/neu 367 139 989 121 171 513 45 9019.0048

Her2/neu.378 Her2/neu 378 10,101 3974

1724 2828 9019.0049

Her2/neu.389 Her2/neu 389 1112

9019.0050 ITGYLYISAWPDSIP Her2/neu.406 Her2/neu 406 96 243 1771 8.5 1369019.0051

Her2/neu.416 Her2/neu 416 — — — 9019.0052

Her2/neu.422 Her2/neu 422 1.3 345 63 33 26 7.1 9019.0053 NLQVIRGRILHNGAYHer2/neu.427 Her2/neu 427 1.9 6879

206 3394 12 9019.0054 RGRILHNGAYSLTLQ Her2/neu.432 Her2/neu 432 2.4 710450 129 2845 5.6 9019.0055 SLTLQGLGISWLGLR Her2/neu.442 Her2/neu 442 93143 110 409 3096 9019.0056 GLGISWLGLRSLREL Her2/neu.447 Her2/neu 447 50122 41 163 3.1 55 9019.0057

Her2/neu.455 Her2/neu 455 7.1 — 646 14

142 9019.0058 GLALIHHNTHLCFVH Her2/neu.464 Her2/neu 464 465 414 171 477277 9019.0059 NTHLCFVHTVPWDQL Her2/neu.471 Her2/neu 471 416 796 207 116178 9019.0060 DECVGEGLACHQICA Her2/neu.502 Her2/neu 502 459 1968 34052337 2097 9019.0061

Her2/neu.518 Her2/neu 518 1915

6742 9019.0062

Her2/neu.532 Her2/neu 532 462 1020 4595 — 540 9019.0063

Her2/neu.543 Her2/neu 543 262 788 9351 812 4861 9019.0064LQGLPREYVNARECL Her2/neu.547 Her2/neu 547 354 1995 211 1144 653 19039019.0065 PSGVKPDLSYMPIWK Her2/neu.601 Her2/neu 601 1832 1578 1525 26889019.0066 ASPLTSIISAVVGIL Her2/neu.648 Her2/neu 648 15 10,905 29 36 71224 9019.0067 ISAVVGILLVVVLGV Her2/neu.655 Her2/neu 655 5153 450 2996 447941 9019.0068 ILLVVVLGVVFGILI Her2/neu.661 Her2/neu 661 — 665 3532 376780 9019.0069 VLGVVFGILIKRRQQ Her2/neu.666 Her2/neu 666 67 2449 177 335101 17 9019.0070 QQKIRKYTMRRLLQE Her2/neu.679 Her2/neu 679 303 3782 5396— 44 9019.0071 IRKYTMRRLLQETEL Her2/neu.682 Her2/neu 682 665 2766 23051050 722 9019.0072

Her2/neu.712 Her2/neu 712 266

3030 176 9019.0073 ETELRKVKVLGSGAF Her2/neu.717 Her2/neu 717 284 19,518246 27 845 101 9019.0074 KVKVLGSGAFGTYYK Her2/neu.722 Her2/neu 722 648.0 204 10,992 77 9019.0075 GENYKIPVAIKYLEE Her2/neu.743 Her2/neu 743491 1055 488 2093

1622.00

Her2/neu.747 Her2/neu 747 1295

125 1178 — 9019.0077 AYVMAGVGSPYVSRL Her2/neu.771 Her2/neu 771 92 164171 596 45 9019.0078 MAGVGSPYVSRLIGI Her2/neu.774 Her2/neu 774 2050 1651352 9019.0079 SRLLGSCLTSTVQLV Her2/neu.783 Her2/neu 783 80 2923 85 13 909.0 9019.0080 TVQLVTQLMPYGCLL Her2/neu.793 Her2/neu 793 164 215 433 43261288 9019.0081 RGRLGGQBLLNWCMQ Her2/neu.814 Her2/neu 814 1059 1412 20299019.0082

Her2/neu.822 Her2/neu 822 944 558 195 1094 380 9019.0083 CMQLAKGMSYLEDVRHer2/neu.826 Her2/neu 826 959 2651 867 1040 116 9019.0084

Her2/neu.832 Her2/neu 832 123 27 957 1357 4315 5496 9019.0085VRLVHRDLAARNVLV Her2/neu.839 Her2/neu 839 59 1503 105 268 561 3569019.0086 HRDLAARVVLVRSPS Her2/neu.843 Her2/neu 843 153 401 765 11,2101332 9019.0087

Her2/neu.848 Her2/neu 848 65 1275 209 197 10,536 118 9019.0088

Her2/neu.865 Her2/neu 865 12 30 14 250 161 664 9019.0008 IKWMALERILRRRFTHer2/neu.886 Her2/neu 886 16 10 37 1075 435 1795 9019.0089

Her2/neu.903 Her2/neu 903 163

2175

9019.0090 IDVWSYGVTVWELMT Her2/neu.903 Her2/neu 903 163 2760 3621 1900546 9019.0091 GVTVWELMTFGAKFV Her2/neu.909 Her2/neu 909 40 377 4.1 2107558 9019.0092 VWELMTTGAKPYDCI Her2/neu.912 Her2/neu 912 36 676 144 4704191 9019.0093 AKPYDGIFAREIPDL Her2/neu.920 Her2/neu 920 116 — — 12,54841 1622.04 ICTIDVYMIMVKCWM Her2/neu.946 Her2/neu 946 — 717 — 955 —9019.0094

Her2/neu.950 Her2/neu 950 1312

7007 274 297 9019.0095 RPBFRELVSEPSBMA Her2/neu.966 Her2/neu 966 26 621838 62 151 309 9019.0096 FRELVSEPSBMARDP Her2/neu.969 Her2/neu 969 20 15022 51 1487 5085 9019.0004

Her2/neu.970 Her2/neu 970 29 35 512 2224 655 1423 9019.0097DGDLGMGAAKGLQSL Her2/neu.1087 Her2/neu 1087 110 254 506 5799 8289019.0099 AKGLQSLPTHDPSFL Her2/neu.1095 Her2/neu 1095 149 194 19 38644459 9019.0000

Her2/neu.1109 Her2/neu 1109 1367 18 25 11,089

197 9019.0100 PEYVNQPDVRPQPPS Her2/neu.1137 Her2/neu 1137 5165 150 —9019.0101 BGPLPAAKPAGATLE Her2/neu.1154 Her2/neu 1154 17,238 — 74639019.0102

Her2/neu.1164 Her2/neu 1164 1812 2792 9019.0103 KDVFAFGGAVENPEYHer2/neu.1182 Her2/neu 1182 1505 — 1577 9019.0173 LPRVGCPALPLPPPPIGFBP2.2 IGFBP2 2 1906 6543 — 9019.0174 ALPLPPPPLLPLLPL IGFBP2.9 IGFBP29 174 18 1.2 20 226 9019.0175 PPPLLPLLPLLLLLL IGFBP2.14 IGFBP2 14 155816 15 16 65 13 9019.0176 LLPLLPLLLLLLGAS IGFBP2.17 IGFBP2 17 119 — 33735 674 964 9019.0177 PLLLLLLGASGGGGG IGFBP2.22 IGFBP2 22 26 19,033 3392144 1122 5982 9019.0178 AEVLFRCEPCTPERL IGFBP2.39 IGFBP2 39 1285 161115,970 9019.0179 PERLAACGPPPVAPP IGFBP2.50 IGFBP2 50 28 5545 163 4353 —9019.0180 PPPVAPPAAVAAVAG IGFBP2.58 IGFBP2 58 178 — 380 91 — 26849019.0181 GARMPCAELVREPGC IGFBP2.73 IGFBP2 73 164 11,639 13,072 — —9019.0015 CAELVREPGCGCCSV IGFBP2.78 IGFBP2 78 12,298 9019.0016

IGFBP2.93 IGFBP2 93 — 9019.0182 ELPLQALVMGEGTCE IGFBP2.121 IGFBP2 1211502 6782 15,638 9019.0017 QALVMGBGTCEKRRD IGFBP2.125 IGFBP2 125 22409019.0183

IGFBP2.157 IGFBP2 157 1153 1196 4623 9019.0184

IGFBP2.169 IGFBP2 169 1021 791 6177 9019.0185 LKSGMKELAVFREKV IGFBP2.184IGFBP2 184 607 — 861 862 34 — 9019.0186 KELAVFREKVTEQHR IGFBP2.190IGFBP2 190 2045 28 9019.0018 ELAVFREKVTEQHRQ IGFBP2.190 IGFBP2 190 50219019.0019 REKVTEQHRQMGKGG IGFBP2.195 IGFBP2 195 2839 9019.0187GKHHLGLEEPKKLRP IGFBP2.209 IGFBP2 209 238 1654 2756 6484 3097 9019.0020KHHLGLEEPKKLRPP IGFBP2.210 IGFBP2 210 4016 9019.0188

IGFBP2.217 IGFBP2 217 1258 806 1122 9019.0189 LDQVLERISTMRLPD IGFBP2.234IGFBP2 234 795 452 25 20 18 5.2 9019.0021 TMRLPDERGPLEHLY IGFBP2.243IGFBP2 243 — 9019.0190 ERGPLEHLYSLHIPS IGFBP2.249 IGFBP2 249 7.7 — 49729 110 18 9019.0191 GLYNLKQCKMSLNGQ IGFBP2.268 IGFBP2 268 923 — 743 28355589 1791 9019.0192 TGKLIQGAPTIRGDP IGFBP2.293 IGFBP2 293 148 9554 40 27405 883 9019.0022

IGFBP2.300 IGFBP2 300 4031 31 1643 2908 — 16,779 9019.0023CHLFYNEQQEARGVH IGFBP2.309 IGFBP2 309

162 1600 3187 — — IC₅₀ nM purified HLA DRB1 DRB1 DRB1 DRB1 DRB1 DRB1DRB3 DRB4 DRB5 Peptide Sequence *0802 *0901 *1101 *1201 *1302 *1501*0101 *0101 *0101 9019.0104 RWCIPWQRLLLTASL 263 4132 191 1558 2326 61 —339 4014 9019.0105 LLTFWNPPTTAKLTI 174 5779 52 4995 245 46 — 2171 319019.0106 TAKLTIESTPFNVAE 1736 4019 5977 2907 35 140 — 53 3350 9019.0107EVLLLVHNLPQHLFG 59 989 40 5.4 0.36 4.7 334 50 1088 9019.0108YSWYKGERVDGNRQI 8432 — 1840 — 533 306 3002 453 1043 9019.0109NRQIIGYVIGTQQAT 46 345 36 4351 990 2.6 — 5.2 2230 9019.0110QYVIGTQQATPGPAY 5877 43 836 6.3 10,223 9019.0111 GPAYSGREIIYPNAS 1549019.0112 GREIIYPNASLLIQN 1959 1355 2209 212 24 49 4035 43 10,6129019.0113 DIGFYTLHVIKSDLV 90 83 174 174 1072 65 14,943 18 564 9019.0114FLTLHVIKSDLVNEE 514 718 6385 616 1340 14 14,343 22 4501 9019.0005LHVIKSDLVNEEATG — — 18,399 — 394 376 — 111 — 9019.0115 KSDLVNEEATGQFRV9963 9019.0116 QFRVYPELPKPSISS 786 9019.0117 KPSISSNNSKPVEDK — 23 975977 — 9019.0118 YLWWVNNQSLPVSPR 119 1912 22 1511 22 116 248 555 9289019.0119 SDSVILNVLYGPDAP 9210 83 351 612 — 9019.0120 LNVLYGPDAPTISPL —218 583 10,798 — 9019.0121 APTISPLNTSYRSGE 3943 125 1706 1047 519019.0122 QYSWFVNGTFQQSTQ 230 14,768 — 11,448 2661 9019.0123QELFIPNITVNNSGS 293 1086 358 1299 26 740 — 3880 3869 9019.0124RTTVTTITVYAEPPK — 2977 83 6287 14,732 9019.0125 TITVYAEPPKPFITS — 232262 — 9255 9019.0126 YLWWVNNQSLPVSPR 243 299 33 1121 1921 227 1088 8612284 9019.0127 SDPVILNVLYGPDDP 5432 161 5699 9262 — 9019.0128SYTYYRPGVNLSLSC 21 1372 776 371 7.2 46 2626 1784 135 9019.0129YSWLIDGNIQQHTQE 1312 9019.0130 NSGLYTCQANNSASG 7338 1024 14,891 100 47179019.0131 RTTVKTITVSAELPK 3763 367 6988 2758 962 29 — 1263 19179019.0132 TITVSAELPKPSISS — 6871 — — 1076 1672 — 10,349 3856 9019.0133KPSISSNNSKPVEDK — 4.1 138 — — 9019.0134 YLWWVNGQSLPVSPR 3195 314 1824295 1348 9019.0135 VCGIQNSVSANRSDP — 921 8979 1051 36 9019.0136QNSVSANRSDPVTLD — 2.7 6657 2105 5816 9019.0137 SSYLSGANLNLSCHS — 3713,028 42 14,095 9019.0138 QYSWRINGIPQQHTQ 151 70 2074 2286 11219019.0139 INGIPQQHTQVLFIA — 256 8287 2700 — 9019.0140 NGTYACFVSNLATGR 70377 2174 2052 30 2351 6963 3247 37 9019.0141 YACFVSNLATGRNNS 1774 4520217 2399 107 1237 — 1569 17 9019.0142 NNSIVKSITVSASGT 184 469 209 13284.4 274 15,808 26 2280 9019.0143 SITVSASGTSPGLSA 3319 592 3110 17415,069 9019.0144 SPGLSAGATVGIMIG — 80 3929 2591 — 1622.05TVGIMIGVLVGVALI — 5637 7770 12,094 384 9019.0006 HQLLCCEVETIRRAY 16,2975607 3471 5585 349 325 — 60 2016 9019.0146 DANLLNDRVLRAMLK 286 9019.0147NDRVLRAMLKAEETC 319 9019.0148 RAMLKAEETCAPSVS — 5017 9145 — 16,12915,733 — 313 1341 9019.0149 FKCVQKEVLPSMRKI 1587 9019.0012QKEVLPSMRKIVATW 1155 1088 58 1121 700 338 — 108 2019 9019.0150LPSMRKIVATWMLEV 1182 137 836 145 8.5 7.1 8449 50 47 9019.0151MRKIVATWMLWVCEE — 356 87 281 17,021 9019.0011 MLEVCEEQKCEEEVF 9019.0152EEEVFPLAMNYLDRF 3414 26 77 72 4444 9019.0153 VFPLAMNYLDRFLSL 259 19,71327 79 5.4 39 239 2.6 2005 9019.0007 DRFLSLEPVKKSRLQ 37 18,378 46 4128 —394 — 399 3.0 9019.0154 DRFLSLEPVKKSRLQ 51 285 1075 227 2.6 9019.0155LEPVKKSRLQLLGAT 1248 221 208 102 731 9019.0156 RLQLLGATCMFVASK 3144 —831 513 227 277 — 421 504 9019.0157 TCMFVASKMKETIPL 61 1031 1113 2423 369019.0158 ASKMKETIPLTAEKL 321 550 1294 2461 2364 1622.01 TIPLTAEKLCIYTDN— — — — — 5360 — 738 253 9019.0159 KLCIYTDNSIRPEEL 16,263 15,928 — — 87925 — 33 4620 9019.0009 DNSIRPEELLQMELL — 3494 4757 87 18,256 9019.0160DNSIRPEELLQMELL 959 9019.0161 PEELLQMELLLVNKL 1091 44 1119 11 28029019.0010 EELLQMELLLVNKLK 1622.06 LLQMELLLVNKLKWN 765 6007 187 1325 4428 — 321 39 9019.0163 MELLLVNKLKWNLAA 368 3090 48 162 4.9 246 — 39 199019.0164 VNKLKWNLAAMTPHD 702 449 1043 116 3.0 356 5867 1839 25199019.0165 KWNLAAMTPHDFIEH 18,625 4876 11,907 194 332 356 — 576 419019.0013 WNLAAMTPHDFIEHF 9019.0166 PHDFIEHFLSKMPEA 820 9019.0167NKQIIRKHAQTFVAL 195 236 34 3.9 2.0 3.3 342 8.7 23 9019.0168AQTFVALCATDVKFI 445 936 276 132 219 97 — 46 18 9019.0169 VKFISNPPSMVAAGS319 752 88 21 24 84 5290 290 826 9019.0170 PPSMVAAGSVVAAVQ 1401 301 16610,090 55 26 — 91 957 9019.0171 VAAVQGLNLRSPNNF 10,850 — 11,089 2018 1711205 — 296 3541 9019.0172 IEALLESSLRQAQQN 12,776 9019.0014EALLESSLRQAQQNM 756 4755 8176 4028 — 9019.0024 LAALCRWGLLLALLP 7049019.0025 WGLLLALLPPGAAST 12 521 21 33 337 1622.02 LLALLPPGAASTQVC 94410,697 17,445 19,806 17 9019.0027 GTDMKLBLPASPETH 14,523 9019.0028RHLYQGCQVVQGNLE 5043 9019.0029 GCQVVQGNLELTYLP 417 9019.0030NLELTYLPTNASLSF 94 3055 30 141 105 23 — 29 189 9019.0031 LTYLPTNASLSFLQD426 4081 213 150 47 132 141 1633 173 9019.0032 IQEVQGYVLIAHNQV 213 302165 3438 103 75 13,508 546 1361 9019.0033 VVLLAHNQVRQVPLQ 300 358 208302 1.9 679 649 124 18 9019.0034 HNQVRQVFLQRLRIV 85 6044 21 42 270 340 —18 173 9019.0035 VRQVPLQRLRIVRGT 2545 9019.0001 GTQLFEDNYALAVLD 9135 4.0450

3744 9019.0036 GDPLNNTTFVTGASP 1030 9019.0037 TTPVTGASPGGLREL 11,154 60517,148 2319 3001 9019.0038

2237 786 4405 2245 16,271 9019.0039

642 343 1183 511 1878 9019.0040 GGVIIQRNPQLCYQD 1720 34 106 351 13,5129019.0041 NPQLCYQDTILWNDI 5074 9019.0042

521 2216 740 441 2009 9019.0043 STDVGSCTLVCPLHN 6781 9019.0044CYGLGMEHLREVRAV — — 3064 18,979 25 690 — 444 3716 9019.0045MEHLREVRAVISANI 95 1345 242 221 23 86 — 81 2.6 9019.0046 LREVRAVTSANIQEF456 5187 641 161 1.5 27 — 163 94 9019.0047

3060 206 635 316 4567 9019.0048

12,191 2905 805 654 — 9019.0049

2322 9019.0050 ITGYLYISAWPDSIP 2573 751 152 270 67 9019.0051

— 9019.0052

148 859 96 486 80 33 — 67 17 9019.0053 NLQVIRGRILHNGAY 119 4217 173 479.9 3.3 4320 446 28 9019.0054 RGRILHNGAYSLTLQ 5077 430 773 40 1.3 5.4358 562 82 9019.0055 SLTLQGLGISWLGLR 6307 1597 50 22 — 9019.0056GLGISWLGLRSLREL 1538 1140 2258 436 3030 5.2 — 154 2048 9019.0057

1075

360 409 16 24 16,162

726 9019.0058 GLALIHHNTHLCFVH 2802 18 164 357 6081 9019.0059NTHLCFVHTVPWDQL 793 175 1431 117 7390 9019.0060 DECVGEGLACHQICA 4976 3236267 1117 12,277 9019.0061

2912 9019.0062

16,162 146 285 1923 7043 9019.0063

254 549 685 2572

9019.0064 LQGLPREYVNARECL 405 670 423 2551 49 9019.0065 PSGVKPDLSYMPIWK1531 9019.0066 ASPLTSIISAVVGIL 3089 118 511 1431 14 59 18,926 1500 2169019.0067 ISAVVGILLVVVLGV 5138 443 6005 3109 — 9019.0068 ILLVVVLGVVFGILI2454 478 7117 11,015 — 9019.0069 VLGVVFGILIKRRQQ 35 — 12 268 17 185 —958 36 9019.0070 QQKIRKYTMRRLLQE

44 2.0 93 340 9019.0071 IRKYTMRRLLQETEL 45 2583 1.6 303 2207 9019.0072

2132 1318 129 368 1223 9019.0073 ETELRKVKVLGSGAF 189 5423 176 2472

205 — 452 1020 9019.0074 KVKVLGSGAFGTYYK 1711 1303 244 1380 13929019.0075 GENYKIPVAIKYLEE 4555 1149 729 84 8353 1622.00

4158 701 611

1305 9019.0077 AYVMAGVGSPYVSRL 1798 402 24 5060

9019.0078 MAGVGSPYVSRLIGI 1508 9019.0079 SRLLGSCLTSTVQLV 634 137 80 4464.7 39 3567 481 392 9019.0080 TVQLVTQLMPYGCLL 11,126 3394 9.4 16 30019019.0081 RGRLGGQBLLNWCMQ 2367 9019.0082

159 2082 799 3359 92 9019.0083 CMQLAKGMSYLEDVR 405 2232 1002 3005 1309019.0084

244 3769 567 2543 467 9019.0085 VRLVHRDLAARNVLV 16 57 1097 298 34259019.0086 HRDLAARVVLVRSPS 1648 316 221 525 302 9019.0087

717 5778 513

15 29 16,142 742 4.9 9019.0088

312 9620 133 65 140 3.3 — 62 3.4 9019.0008 IKWMALERILRRRFT 515 9282 136241 1118 11 — 340 3.3 9019.0089

1150 16,270 2726 618 5301 9019.0090 IDVWSYGVTVWELMT 2639 467 1879 — 54259019.0091 GVTVWELMTFGAKFV 315 8368 20 1171 34 9019.0092 VWELMTTGAKPYDCI1952 1553 245 3565 65 9019.0093 AKPYDGIFAREIPDL — 19,575 6957 — 30411622.04 ICTIDVYMIMVKCWM 1480 — 2652 — — 9019.0094

172 395 1303 607 716 9019.0095 RPBFRELVSEPSBMA 376 2321 125 1779 12,182348 — 351 26 9019.0096 FRELVSEPSBMARDP 1714 — 331 — — 124 4537 1282 3809019.0004

790 1401 40

240 1405 901 227 45 9019.0097 DGDLGMGAAKGLQSL 2233 3234 1634 896 21419019.0099 AKGLQSLPTHDPSFL 6806 3991 1665 572 — 9019.0000

94 380 531 9019.0100 PEYVNQPDVRPQPPS 1714 9019.0101 BGPLPAAKPAGATLE10,247 9019.0102

9019.0103 KDVFAFGGAVENPEY 16,146 9019.0173 LPRVGCPALPLPPPP 89369019.0174 ALPLPPPPLLPLLPL 1087 41

1521 263 10

9019.0175 PPPLLPLLPLLLLLL 86 307 121 23 1322 5.0 16,239 2.1 1219019.0176 LLPLLPLLLLLLGAS 213 5893 458 320 2022 182 — 19 2390 9019.0177PLLLLLLGASGGGGG

— 12,919 19,255 2161 252 — 216 — 9019.0178 AEVLFRCEPCTPERL 57749019.0179 PERLAACGPPPVAPP 3541 3618 4054 4455 19,615 9019.0180PPPVAPPAAVAAVAG — 317 — — 237 1378 —

— 9019.0181 GARMPCAELVREPGC 2968 16,279 — 4697 — 9019.0015CAELVREPGCGCCSV 9019.0016

9019.0182 ELPLQALVMGEGTCE 9167 9019.0017 QALVMGBGTCEKRRD 9019.0183

2518 9019.0184

5686 9019.0185 LKSGMKELAVFREKV 260 — 163 1768 4974 91 — 417 8439019.0186 KELAVFREKVTEQHR 9019.0018 ELAVFREKVTEQHRQ 9019.0019REKVTEQHRQMGKGG 9019.0187 GKHHLGLEEPKKLRP 16,789 3029

1378 6390 9019.0020 KHHLGLEEPKKLRPP 9019.0188

1213 9019.0189 LDQVLERISTMRLPD 1607 52 467 314 1052 84 — 367 2239019.0021 TMRLPDERGPLEHLY 9019.0190 ERGPLEHLYSLHIPS 9361

1995 50 1149 23 — 1648 4000 9019.0191 GLYNLKQCKMSLNGQ 204 9434 2660 413561 223 — 1613 5609 9019.0192 TGKLIQGAPTIRGDP 5169 709 7989 2091 9271783 2191 405 36 9019.0022

— — — 16,917 1674 3669 191 2510 — 9019.0023 CHLFYNEQQEARGVH — — — 1855145 — 18,902 5310 — — indicates binding affinity ≧ 20.000 nM.

indicates data missing or illegible when filed

TABLE II Breast/Ovarian HLA-DR Supertype CandidatesIC₅₀ nM to purified HLA Peptide DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 No.Sequence Source Protein Position

9019.0105 LLTFWNPPTTAKLTI CEA.24 CEA 24 6.9 16,313 273 52 258 3.7 1749019.0106 TAKLTIESTPFNVAE CEA.33 CEA 33 72 613 106 41 383 70 17369019.0107 EVLILVHNLPQHLFQ CEA.50 CEA 50 2.7 830 3.4 1.7 30 5.4 199019.0108 YSWYKGERVDGNRQI CEA.65 CEA 65 511 — 34 585 360 866 8429019.0109

CEA.76 CEA 76 216 — 108 1.5 129 46 16 9019.0112 GREIIYPNASLLIQN CEA.97CEA 97 62 433 251 88 550 29

9019.0113 DTQFYTLHVEKSDLV CEA.116 CEA 116 64 964 84 260 95 23 709019.0114

CEA.119 CEA 119 101 80

160 41 46

9019.0118

CEA.176 CEA 176 2.4 100 872 203 80 17 119 9019.0123 QELFIPNITVNNSGSCEA.282 CEA 282 147 644 25 227 379 1658 293 9019.0126 YLWWVNNQSLPVSPRCEA.354 CEA 354 1128 214 12 248 83 28 243 9019.0128 SYTYYRPGVNLSLSCCEA.423 CEA 423 1.6 4425 6.8 4036 300 5.4 21 9019.0131

CEA.488 CEA 488 80

84 11 1.5 9019.0140 NGTYACFVSNLATQR CEA.650 CEA 650 539 818 11 558 30 20

9019.0141 YACFVSNLATGRNNS CEA.653 CEA 653 183 774 225 41 327 531 11749019.0142

CEA.665 CEA 665 34 103 43 1.8 128 34 134 9019.0006 HQLLCCEVETTRRAYCyclin D1.3 Cyclin D1 3 953 21 740 256 4209 11,553 16,297 9019.0012

Cyclin D1.49 Cyclin D1 49

111 12,162 1102 3720 451 1155 9019.0150 LPSNRKIVATWMLEV Cyclin D1.53Cyclin D1 53 8.5 820 238 28 123 4.6 1182 9019.0153 VFPLAMNYLDRFLSLCyclin D1.77 Cyclin D1 77 146 107 2332 1567 609 3332 259 9019.0007DRFLSLEPVKKSRLQ Cyclin D1.86 Cyclin D1 86 16 290 18 61 159 1057 379019.0156 RLQLLGATCMFVASK Cyclin D1.98 Cyclin D1 98 12 — 91 533 1439 4583164 1622.06 LLQMELLLVNKLKWN Cyclin D1.137 Cyclin D1 137 39 — 3539 6.4332 2196 735 9019.0163 MELLLVNKLKWNLAA Cyclin D1.140 Cyclin D1 140 469006 177 191 2411 1797 358 9019.0164 VNKLKWNLAAMTPHD Cyclin D1.145Cyclin D1 145 46 1419 88 781 747 382 712 9019.0165

Cyclin D1.149 Cyclin D1 149 33 6249 3405 553 122 1101 18,625 9019.0167NKQTRKIIAQTFVAL Cyclin D1.174 Cyclin D1 174 4.7 561 6.5 23 25 4.4 1359019.0168 AQIIVALCATDVRFI Cyclin D1.182 Cyclin D1 182 5.4 551 26 133 5513 445 9019.0169

Cyclin D1.193 Cyclin D1 193 6.7 4128 12 18 117 304 318 9019.0170PPSMVAAGSVVAAVQ Cyclin D1.199 Cyclin D1 199 6.8 — 12 26 3718 133 14019019.0171 VAAVQGLNLRSPNNF Cyclin D1.209 Cyclin D1 209 44 18,558 226 5324248 161 10,850 9019.0030 NLELTYLPTNASLSF Her2/neu.59 Her2/neu 59 4.97356 6.2 2.7 38 7.2 94 9019.0031 LTYLPTNASLSELQD Her2/neu.62 Her2/neu 629.7

19 16 60 15 426 9019.0032

Her2/neu.77 Her2/neu 77 57 7763 111 178 102 35 213 9019.0033YVLIAHNQVRQVPLQ Her2/neu.83 Her2/neu 83 28 454 53 104 1185 92 3309019.0034 HNQVRQVPLQRLRIV Her2/neu.88 Her2/neu 88

971 840 78 1303 80 15 9019.0035

Her2/neu.347 Her2/neu 347 9.4 2040

12

9.7

9019.0046 LREVRAVTSANIQEF Her2/neu.350 Her2/neu 350 17 3913 43 8.2 50 12456 9019.0052 LSVFQNLQVIRGRIL Her2/neu.422 Her2/neu 422 1.3 345 6.3 3326 7.1 148 9019.0054

Her2/neu.432 Her2/neu 432 2.4 713 480 129 2845 5.6 5477 9019.0057LRSLRELGSGLALIH Her2/neu.455 Her2/neu 455 7.1 — 896 14 603 142 14759019.0009

Her2/neu.666 Her2/neu 666 87 2449 177 555 101 17 15 9019.0079SRLLGPCLTSTVQLV Her2/neu.783 Her2/neu 783 80 2923 85 13 90 9.0 6549019.0087 PIKWMALESILRRRF Her2/neu.885 Her2/neu 885 12 30 14 250 161 664112 9019.0003 IKWMALESILRRRFT Her2/neu.886 Her2/neu 886 16 10 37 1075435 1795 515 9019.0004 FSRMARDPQRFVVIQ Her2/neu.976 Her2/neu 976 29 35512 2224 855 1423 798 9019.0174 ALPLPPPPLLPLLPL IGFBP2.9 IGFBP2 9 174 —18 4.2 20 336 1087 9019.0175 PPPLLPLLPLLLLLL IGFBP2.14 IGFBP2 14 15 581615 16 65 13 86 9019.0176 LLPLLPLLLLLLGAS IGFBP2.17 IGFBP2 17 1.9 — 33735 674 964 213 9019.0177 PLLLLLLGASGGGGG IGFBP2.22 IGFBP2 22 20

330 2144 1422

9019.0180

IGFBP2.58 IGFBP2 58 178 — 380 81 — 2684 — 9019.0185

IGFBP2.184 IGFBP2 184 687 — 881 862 34 — 260 9019.0189 LDQVLERISTMRLPDIGFBP2.234 IGFBP2 234 735 452 25 29 18 5.2 1997 9019.0190EROPLEHLYSLHIPN IGFBP2.249 IGFBP2 249 7.5 — 497 29 110 18 9361 9019.0191GLYNLKQCKMSLNGQ IGFBP2.268 IGFBP2 268 923 — 743 2835 5583 1791 2049019.0192 TGKLIQGAPTIRGDP IGFBP2.293 IGFBP2 293 165 9554 40 27 608 8835160 IC₅₀ nM to purified HLA Peptide DRB1 DRB1 DRB1 DRB1 DRB1 DRB3 DRB4DRB5 Total No. Sequence

XRN 9019.0105 LLTFWNPPTTAKLTI 5779 52 4995 245 46 — 2171 31 10 9019.0106TAKLTIESTPFNVAE 4019 5977 2947 35 140 — 53 3350 9 9019.0107EVLILVHNLPQHLFQ 989 40 5.4 0.36 4.7 334 30 1165 14 9019.0108YSWYKGERVDGNRQI — 1540 — 533 306 3002 453 1043 8 9019.0109

345 36 4351 990 2.6 — 5.2 2292 11 9019.0112 GREIIYPNASLLIQN 1399 2209212 24 49 4035 43 10,612 10 9019.0113 DTQFYTLHVEKSDLV 83 174 174 1072 6514,943 18 564 13 9019.0114

718 6385 616 1340 14

4501 11 9019.0118

1912 22 1511 22 116 248 555 328 13 9019.0123 QELFIPNITVNNSGS 1086 3581249 26 740 — 3889 3869 9 9019.0126 YLWWVNNQSLPVSPR 299 33 1121 1921 2271081 861 2284 10 9019.0128 SYTYYRPGVNLSLSC 1372 776 378 7.2 46 2621 1784135 10 9019.0131

1917 7 9019.0140 NGTYACFVSNLATQR 377 2174 2052 30 2351 6065 3247 37 109019.0141 YACFVSNLATGRNNS 4520 217 2339 107 1237 — 1509 17 9 9019.0142

469 200 1328 4.4 274 15,808 26 2280 12 9019.0006 HQLLCCEVETTRRAY 56073471 5545 349 325 — 60 2016 7 9019.0012

1066 36 1121 700 336 — 106 2013 8 9019.0150 LPSNRKIVATWMLEV 137 886 1458.5 7.1 8449 50 47 13 9019.0153 VFPLAMNYLDRFLSL 19,713 27 78 5.4 39 2392.6 2005 10 9019.0007 DRFLSLEPVKKSRLQ 18,378 46 4128 — 394 — 359 3.0 109019.0156 RLQLLGATCMFVASK — 831 713 227 277 — 421 504 10 1622.06LLQMELLLVNKLKWN 6097 187 1305 44 28 — 321 39 9 9019.0163 MELLLVNKLKWNLAA3090 46 162 4.9 246 — 30 13 10 9019.0164 VNKLKWNLAAMTPHD 449 1043 1153.0 356 586 1839 2519 10 9019.0165

4876 11,907 194 332 356 — 576 41 8 9019.0167 NKQTRKIIAQTFVAL 236 34 3.91.2 3.3 342 8.7 23 15 9019.0168 AQIIVALCATDVRFI 536 276 132 219 97 — 4618 14 9019.0169

752 88 21 24 84 5299 290 826 13 9019.0170 PPSMVAAGSVVAAVQ 301

10,090 55 26 — 91 957 10 9019.0171 VAAVQGLNLRSPNNF — 11,089 2018 1711205 — 296 3541 6 9019.0030 NLELTYLPTNASLSF 3055 30 141 105 23 — 29 18912 9019.0031 LTYLPTNASLSELQD 4061 213 157 47 112 141 1631 173 129019.0032

302 165 3438 103 75 13,508 546 1361 11 9019.0033 YVLIAHNQVRQVPLQ 358 208302 1.9 679 649 124 18 14 9019.0034 HNQVRQVPLQRLRIV 6644 21 42 270 340 —18 173 12 9019.0035

221

84 81

12 9019.0046 LREVRAVTSANIQEF 5187 661 162 1.5 27 — 163 94 12 9019.0052LSVFQNLQVIRGRIL 859 9.6 488 80 33 — 67 17 14 9019.0054

430 773 40 1.3 5.4 358 562 82 13 9019.0057 LRSLRELGSGLALIH 594 309 49516 24 16,142 549 726 12 9019.0009

— 12

17 165 —

36 12 9019.0079 SRLLGPCLTSTVQLV 137 80 445 4.7 39 3567 481 392 139019.0087 PIKWMALESILRRRF 3620 133 66

3.3 — 62

13 9019.0003 IKWMALESILRRRFT 9282 136 241 1118 11 — 340 3.3 10 9019.0004FSRMARDPQRFVVIQ 1481 49 6367 240 1404 — 227 45 10 9019.0174ALPLPPPPLLPLLPL — 41 1337 1524 263 16,239 40 9665 8 9019.0175PPPLLPLLPLLLLLL 307 121 23 1322 5.0 — 2.1 121 12 9019.0176LLPLLPLLLLLLGAS 5803 498 120 2002 182 — 19 2390 10 9019.0177PLLLLLLGASGGGGG — 12,818 18,255 2164 252 — 314 — 4 9019.0180

317 — — 237 1371 — 1888 — 5 9019.0185

— 163 1768 4974 91 — 417 843 9 9019.0189 LDQVLERISTMRLPD 52 467 314 105284 — 367 223 12 9019.0190 EROPLEHLYSLHIPN 638 1993 50 1149 23 — 16484000 8 9019.0191 GLYNLKQCKMSLNGQ 9434 2000 413 501 223 — 1613 5009 69019.0192 TGKLIQGAPTIRGDP 1709 7939 2991 927 1731 2191 465 36 9 —indicates binding affinity ≧ 20.000 nM.

indicates data missing or illegible when filed

TABLE III Vaccine Candidates DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1DRB1 DRB1 DRB1 DRB1 DRB3 DRB4 DRB5 EPITOPE % *0101 *0301 *0401 *0404*0405 *0701 *0802 *0901 *1101 *1201 *1302 *1501 *0101 *0101 *0101HER2/NEU.59 15 X X X X X X X X X X X X HER2/NEU.885 25 X X X X X X X X XX X X CEA.24 17 X X X X X X X X X X CEA.653 25 X X X X X X X IGFBP2.1719 X X X X X X X X IGFBP2.249 23 X X X X X X X CYCLIND1.53 13 X X X X XX X X X X X CYCLIND1.199 13 X X X X X X X X X % = Percent of patientswith responses

1. (canceled)
 2. A method for eliciting an immune response to a tumorantigen within a mammal, wherein said method comprises administering avaccine composition to said mammal, wherein said vaccine compositioncomprises an adjuvant and an isolated HLA-DR binding peptide having thesequence set forth in SEQ ID NO:145, and further comprises at least oneadditional peptide having the sequence set forth in SEQ ID NO:89 or SEQID NO:90.
 3. The method of claim 2, wherein said mammal is a human. 4.The method of claim 2, wherein said vaccine composition furthercomprises a HTL-inducing peptide.
 5. The method of claim 2, wherein saidvaccine composition further comprises a CTL peptide.
 6. The method ofclaim 2, wherein said vaccine composition further comprises a lipid. 7.The method of claim 2, wherein said vaccine composition furthercomprises a liposome.
 8. The method of claim 2, wherein said vaccinecomposition further comprises at least two Carcinoembryonic Antigen(CEA) HTL peptides, at least two Cyclin D1 HTL peptides, and at leasttwo Insulin Growth Factor Binding Protein 2 (IGFBP-2) HTL peptides. 9.The method of claim 1, wherein said at least one additional peptide hasthe sequence set forth in SEQ ID NO:89.
 10. The method of claim 1,wherein said at least one additional peptide has the sequence set forthin SEQ ID NO:90.